JP2001190280A - Collagen-binding physiologically active polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject polypeptide where the activity of the physiologically active peptide is retained and the activity to bind to collagen is imparted. SOLUTION: This polypeptide is a hybrid polypeptide having both activities, i.e., collagen binding activity and physiological activity, wherein a physiologically active peptide is linked to a fibronectin-derived peptide. A collagen matrix is obtained by complexing the above polypeptide with collagen. The above polypeptide is useful as a drug delivery system(DDS) for the relevant physiologically active peptide. The above-mentioned functionally modified collagen matrix is useful as a new biomaterial for tissue regeneration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to hybrid polypeptides, and more particularly, to fibronectin (FN).
A collagen-binding bioactive polypeptide comprising a peptide derived from a bioactive peptide and a bioactive peptide linked by a genetic engineering technique, wherein the polypeptide has both collagen-binding activity and bioactivity. The present invention relates to a biomaterial obtained by compounding collagen and a polypeptide derived from collagen.

[0002]

2. Description of the Related Art Many bioactive peptides such as cytokines and cell growth factors are expected to be used as pharmaceuticals. For example, basic fibroblast growth factor (bFGF) is a cell against mesoderm and neuroectoderm derived proliferation and differentiation promoting activity, vascular endothelial cells, fibroblasts, vascular smooth muscle cells, cells against astroglial cells A polypeptide having various biological activities such as growth activity, cell migration activity and angiogenesis activity, and is widely used as a growth factor for cultured cells (Journal of Cell Biology, No. 1).
09, No. 1, pages 1 to 6 (1989)). Due to its cell proliferation activity, cell migration activity, and angiogenesis activity, bFGF is expected to be used as a wound healing agent (RAF Clark
Ed., The Molecular and Cellular Biology of Wound Re
pair, 2nd edition, Chapter 6, pp. 237-248 (198
8), Plenum Publishing, New York). Epidermal growth factor (E
GF) is also expected to be used as a wound healing agent because of its promotion of proliferation of epithelial cells, vascular endothelial cells, fibroblasts, etc. and angiogenesis activity (Growth Facto, edited by Ziegler et al.).
rs and Wound Healing, Part 3, Chapter 12, 206-2
28 (1997), Springer-Verlag, New York).

[0003] However, bioactive peptides generally have poor stability in vivo and are difficult to locally retain and sustainedly release, so that they have low effects on target tissues and have side effects at other sites. For this reason, there are many concerns about practicality, and an efficient drug delivery system (DDS) is eagerly desired.

In order to solve this problem, many attempts have been made to ensure sustained release in vivo by mixing a bioactive peptide with a biocompatible polymer. Among the biocompatible polymers, collagen is a protein widely used in the medical field as a biomaterial for repairing biological damage or as a drug carrier, and has excellent biocompatibility .

A technique for controlling the sustained release of a bioactive peptide by mixing it with collagen has been described.
For example, Japanese Patent No. 2641755, Japanese Unexamined Patent Publication No. 5-43453
No. 6,086,045. Japanese Patent Publication No. 18865/1992 discloses a collagen matrix containing a cell growth factor, which is obtained by mixing a cell growth factor with collagen and then subjecting the mixture to thermal dehydration crosslinking. Furthermore, for the purpose of sustained release of the bioactive peptide, a method for preparing a sustained-release matrix in which heparin, a bioactive peptide having heparin-binding properties, and collagen are mixed (Japanese Patent Application Laid-Open Nos. 8-253429 and 8- 3
No. 25160). It is presumed that by mixing such materials, sustained release of the heparin-binding bioactive peptide can be achieved. However, the method using both heparin and a bioactive peptide has a limitation that it cannot be applied to a bioactive peptide having no heparin binding property, in addition to the problem of anticoagulant action of heparin.

[0006] A method has been reported in which interferon is mixed and kneaded with high-concentration collagen, and air-dried collagen pellets are embedded in a living body to increase the stability and local retention (Fujioka et al.). , Journa
l of Controlled Release Vol. 33, 307-315
P. (1995)).

[0007] Alternatively, an antithrombotic material in which heparin and collagen are chemically bonded directly (Raghunath et al., Journal of
Biomedical Materials Research, Vol. 17, 613
621 (1983); Nimni et al., Journal of Biome.
dical Materials Research, Volume 21, 741-77
1 (1987)). As another method, a method of chemically bonding a physiologically active peptide and collagen with glutaraldehyde or the like has been attempted.

As described above, efficient DDS is desired for many bioactive peptides, and collagen is expected as a carrier for enhancing the sustained release of bioactive peptides. The affinity is generally low, and it is impossible to stably hold the bioactive peptide on the collagen matrix for a long time by simply mixing the two. Further, the method of chemically binding a physiologically active peptide to collagen has a problem that its biological activity is lost or reduced.

[0009] In contrast, in recent years, bioactive peptides have been hybridized with other peptides by genetic engineering,
Development of a technique for achieving targeting of a physiologically active peptide has been promoted. That is, it is possible to express a hybrid polypeptide in Escherichia coli or the like by using a genetic recombination technique, but the expressed hybrid polypeptide does not always show the original activity, and a desired activity, particularly a physiological activity Can be difficult to obtain.

Japanese Patent Application Laid-Open No. 5-178897 discloses that fibronectin (F
A method for producing a functional polypeptide in which the cell adhesion domain sequence of N) is hybridized with bFGF is disclosed. This functional polypeptide adheres to cells and exhibits cell growth activity, but cannot be expected to have a long-term sustained release effect or local retention because it is directly bound to cells. It also describes that the activity of the functional polypeptide as bFGF is significantly reduced.

[0011] The target of the sustained-release effect via the extracellular matrix is a hybrid poly-protein composed of a collagen-binding decapeptide derived from bovine von Willebrand factor and transforming growth factor β (TGF-β). A peptide, collagen targeting TGF-β, has been reported (USP 5,800,811, Tu
an et al., Connective Tissue Research, Vol. 34, No. 1,
1-9 (1996); Han et al., Protein Expression a.
nd Purification, Vol. 11, pp. 169-178 (19
97), Gordon et al., Human Gene Therapy, Vol. 8, 1385.
1394 (1997)). However, many of the recombinant proteins recovered from the inclusion bodies produced by E. coli have impaired their collagen binding activities and have also been found to have reduced physiological activities. In particular, the hybrid polypeptide in which the collagen-binding activity remained remained bound to collagen, and then cells could be captured.
No proliferative activity of -β was reported.
The purpose of local retention and sustained release of -proliferation activity has not been achieved. Therefore, the collagen-binding decapeptide of von Willebrand factor cannot be said to be appropriate as the collagen-binding peptide portion of the collagen-binding bioactive polypeptide that is a hybrid polypeptide.

On the other hand, Nishi et al., Clostridium histolyticum (Clostridi
um histolyticum), a collagen-binding polypeptide derived from collagenase, and a collagen-binding cell growth factor which is a hybrid polypeptide of bFGF or EGF. It was described that they did not have sufficient collagen binding activity because they could not bind to an incubator or many collagen materials (P
roc. Nat. Acad. of Sci. USA, Vol. 95, 7018-
7023 (1998)). Naturally, it is not possible to show cell proliferation activity after binding to collagen material. Furthermore, the collagen-binding cell growth factor EG
F activity has been found to decrease. Also, immunologically it is clear that polypeptides derived from bacterial collagenase are not suitable for human tissue regeneration.

When producing a hybrid polypeptide using a gene recombination technique in consideration of industrial production, production of a prokaryote (for example, a bacterium) or yeast, particularly expression of an Escherichia coli system is preferred for cost reduction. Realization is an important issue. However, in E. coli expression, it is often difficult for the obtained polypeptide to exhibit the original activity or to efficiently produce a polypeptide having the desired activity.
One of the major problems in E. coli-based expression is the inability to obtain sufficient soluble polypeptide. this is,
There are cases where the polypeptide becomes insoluble as an inclusion body and cases where it is degraded immediately after synthesis. It is thought that the high expression of the exogenous polypeptide puts stress on Escherichia coli and a mechanism to eliminate it works. The mechanism of inclusion formation is still largely unknown, and there is no perfect solution to prevent it. It is also effective to solubilize the inclusion body with a strong protein denaturant and then regenerate the target protein.However, since the denaturation process is performed once, the three-dimensional structure of the target polypeptide is not correctly regenerated in the end. In many cases, it does not show any significant biological activity.

As a method for expressing a general-purpose hybrid polypeptide, there is known a method in which a soluble protein having an activity is efficiently produced in an Escherichia coli expression system and can be regenerated even if an inclusion body is formed. Polypeptide partners for hybrids used in this method include thioredoxin, protein A, chloramphenicol acetyl transferase, lac repressor, galactose binding protein, maltose binding protein (MB
P), glutathione-S-transferase (GS
T), β-galactosidase and the like, and many expression vectors are already commercially available.

When these polypeptide partners are used, there is a relatively high possibility of obtaining a hybrid polypeptide which is soluble and retains its activity, but none of them shows collagen binding properties. And in the case of using a hybrid polypeptide other than these partners,
The possibility of expression and production is low, and it is expected that the possibility of obtaining a protein maintaining its activity will be even lower. That is, in order to prove the feasibility of an active hybrid polypeptide, it is necessary to produce a hybrid polypeptide using a partner and demonstrate its activity.

It is conceivable to use an FN collagen-binding region as a collagen (gelatin) -binding polypeptide partner. For example, it has been proposed to select an FN-derived polypeptide as a tag for the purification of a useful protein. Specifically, a vector for expressing a polypeptide in which a useful protein is linked to a gelatin-binding region of FN and a method for purifying the useful protein (US Pat. No. 5,342,762 and US Pat. No. 5,460,955)
), A polypeptide in which a collagen-binding portion of FN is bound to another polypeptide or a therapeutic agent, and a method for purifying the same (Japanese Patent Application Laid-Open No. 62-89699).

In the above-mentioned US patent, the sequence-limited gelatin-binding region of rat FN is claimed, and the affinity of the FN-derived polypeptide (purified tag) for collagen / gelatin is compared with that of the FN-derived polypeptide and useful protein. After capturing the hybrid polypeptide with the above, only the useful protein portion is recovered by subsequent cleavage with a specific protease or the like.

Specifically, at the amino terminus of the gelatin-binding region of FN, a prepro sequence or leader signal peptide of FN which is a 32-residue amino acid sequence that secretes a recombinant polypeptide from insect cells or animal cells into a medium. Example by adding sequence, production in insect cells by baculovirus expression system, purification or COS
It is produced and purified using animal cells such as cells as hosts. More specifically, a cleavage site by trypsin is located on the amino-terminal side of the gelatin-binding region of the FN, a useful protein is located on the amino-terminal side, followed by a factor XIIIa site, FN prepro sequence or leader signal peptide sequence is added to the side.

However, the above-mentioned US Patent Publication does not disclose at all whether or not there is a physiological activity of a useful protein which is a portion other than the FN-derived polypeptide in the hybrid polypeptide. That is, as an example of a method for purifying a useful protein, a homology unit of FN is used.
I-12 (12th type I homology unit), FN homology unit I-9 (9th type I homology unit)
And the various parts of III-1 (the first type III homology unit) or human Factor IX have been expressed and purified, but these polypeptides are not the bioactive peptides described in the present invention and have no bioactivity. Therefore, the activity, function, and use of the hybrid polypeptide itself when the useful protein is a physiologically active peptide are not described at all. That is, there is no reference to uses such as human cell proliferation, tissue regeneration, and organ regeneration, and even if the use is considered, the above configuration is inappropriate.

That is, as described above, the useful protein is F
In the ligation located on the amino-terminal side of the gelatin-binding region of N, a cleavage site by trypsin is present, or even if a protease-cleaving sequence is newly inserted therein, an extra site such as a protease recognition sequence is added to the carboxyl-terminal of the useful protein. When a useful protein is a bioactive peptide, at least one amino acid sequence remains after cleavage by the protease, resulting in a decrease or disappearance of the bioactivity.

If the purpose is merely purification, it is possible to obtain a useful protein containing no extra sequence by cleaving with an endoprotease such as trypsin in vitro or in vitro, followed by degradation with carboxypeptidase B or the like. is there. However, unless treated by such a procedure, the carboxyl terminus of a useful protein will contain an extra amino sequence after cleavage by an endoprotease, causing a reduction or disappearance of the activity of the useful protein. Therefore, it is not suitable for the purpose of directly using a collagen-binding bioactive polypeptide which is a hybrid polypeptide as in the present invention.

Further, the polypeptide having the above constitution is
In particular, production of a hybrid peptide in a prokaryote such as Escherichia coli causes a decrease or disappearance of collagen binding, or a decrease or disappearance of a biological activity when the useful protein is a bioactive peptide. Alternatively, the recovery of the active polypeptide from the inclusion bodies is extremely low or impossible. That is, it is relatively easy to produce an animal-derived polypeptide using an animal cell or an insect cell as a host while maintaining its function, but it is very difficult to realize it in a prokaryote (such as a bacterium) or yeast. In particular, production of a recombinant polypeptide by a prokaryote such as Escherichia coli is very advantageous for cost reduction, but is very difficult, and its ingenuity is required for realizing it.

Also, Japanese Patent Application Laid-Open No. 62-89699 discloses that
Although sequence comprising a polypeptide composed amino sequence from Thr ³⁷⁹ of human FN to Val ^{44 5} are shown, it can not be said polypeptide shown herein is useful as a partner of the hybrid polypeptide. As will be described later, the sequence used in the examples of this publication is a sequence that can be produced by genetic engineering, unlike the sequence obtained by protease treatment, and the sequence used is a polypeptide partner with a bioactive peptide. The hybrid polypeptide showed little collagen binding. In addition, the physiological activity decreased or disappeared.

Another technique for targeting a functional protein to the extracellular matrix is a chimeric protein comprising a heterologous protein or peptide amino acid chain inserted between the N-terminal 70 kDa region and the C-terminal 37 kDa region of the FN molecule. It has been proposed in JP-A-8-140677.
There are two main practical obstacles to the present invention. That is, F
Since it is inserted between N molecules, it is difficult to maintain a higher-order structure, and there is an example where it is expressed in animal cells to maintain the function of some functional proteins, but its versatility is low. In addition, the molecular weight is large,
Escherichia coli (Escherichia coli) because it is insoluble in water
Industrial production is impossible. Even if the function can be maintained, it is considered that the only use is gene therapy in which gene expression is performed directly in human cells.

[0025]

Although it has been proposed to express a hybrid polypeptide in Escherichia coli or the like using a gene recombination technique, an active hybrid polypeptide cannot be obtained in many cases. An object of the present invention is to provide a collagen-binding bioactive polypeptide to which collagen-binding activity has been imparted by genetic engineering without losing the activity of the bioactive peptide. An object of the present invention is to provide a biomaterial (bioactive peptide-complexed collagen) in which the collagen-binding bioactive polypeptide and a collagen-derived polypeptide are complexed to achieve a long-term and adjustable sustained release function.

[0026]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors linked the collagen-binding domain of fibronectin (FN) to a bioactive peptide by a genetic engineering technique. The activity of the peptide is maintained, and it has been proved that it is possible to produce a collagen-binding bioactive polypeptide to which collagen-binding activity has been imparted. The present invention was devised by devising a biomaterial (bioactive peptide-complexed collagen) in which the above was complexed.
That is, the object of the present invention is solved by the following 1) to 15).

1) A polypeptide in which a peptide derived from fibronectin and a physiologically active peptide are linked, and which has both collagen-binding activity and physiological activity. 2) The collagen-binding bioactive polypeptide according to 1) above, wherein the bioactive peptide is linked to the carboxyl terminus of a fibronectin-derived peptide.

3) The fibronectin-derived peptide is obtained by digestion of fibronectin with a protease and comprises an amino acid sequence constituting a collagen binding domain, or a sequence homologous to the sequence, or a partial substitution or deletion thereof. The collagen-binding physiologically active polypeptide according to the above 1) or 2), which comprises a sequence that is lost, inserted, or adducted and has collagen-binding properties. The fibronectin is preferably human fibronectin. Those obtained by limited or spontaneous degradation of human fibronectin by a protease are preferred. Preferably, the protease is a combination of both plasmin and chymotrypsin.

More specifically, 4) the fibronectin-derived peptide is an amino acid sequence constituting the polypeptide from Ala ²⁶⁰ to Trp ⁵⁹⁹ of human fibronectin, or is homologous or partially substituted with the sequence. The collagen-binding bioactive polypeptide according to any one of 1) to 3) above, which comprises a sequence which is a deletion, insertion, or adduct. Here, collagen binding is synonymous with gelatin binding.

5) The physiologically active peptide is a cytokine,
The collagen-binding bioactive polypeptide according to any one of 1) to 4) above, which is a cell growth factor. As the cell growth factor, the fibroblast growth factor (FGF) family or the epidermal growth factor (EGF) family is suitable.

6) The collagen-binding bioactive polypeptide according to 1) to 5) above, wherein the bioactive peptide is linked to the fibronectin-derived peptide by a genetic engineering technique. 7) The bioactive peptide is linked to the fibronectin-derived peptide, preferably at the carboxyl terminus, by genetic engineering techniques, and the bacteria, preferably Esherichia c
The collagen-binding physiologically active polypeptide according to any one of 1 to 6), which is produced in oli (Escherichia coli). In the above, an amino acid or a peptide may be inserted as a spacer at the linking site between the physiologically active peptide and the peptide derived from the fibronectin. In this case, it is preferable that either the spacer or the spacer and a sequence adjacent thereto include a protease recognition sequence. The protease recognition sequence is preferably an enterokinase recognition sequence.

(8) The collagen-binding bioactive polypeptide according to any one of (1) to (7) above, wherein the collagen-binding bioactive polypeptide is water-soluble.

9) The collagen-binding physiologically active polypeptide according to any one of 1) to 8) above, which exhibits physiological activity after binding to collagen while maintaining the binding or being released gradually. 10) A local maintenance agent, sustained release agent or bioactivity imparting agent for a bioactive polypeptide containing the collagen-binding bioactive polypeptide according to any one of 1) to 9) above.

11) A biomaterial containing a physiologically active peptide-complexed collagen in which a collagen-binding physiologically active polypeptide and a collagen-derived polypeptide are complexed. 12) The biomaterial according to 11), wherein the collagen-binding bioactive polypeptide is the collagen-binding bioactive polypeptide according to any one of 1) to 9). It is possible to provide a method for imparting the biological activity of proliferation, differentiation, regeneration or promotion of substance production to cells, tissues and organs using the biomaterial described in the above item 11) or 12). 13) A local maintenance agent, sustained release agent or bioactivity imparting agent for a bioactive polypeptide containing the biomaterial according to the above item 11) or 12).

14) A gene encoding the collagen-binding physiologically active polypeptide according to any one of 1) to 9) above. A recombinant vector containing a gene encoding the collagen-binding bioactive polypeptide according to any one of the above 1) to 9). A transformant containing a gene encoding the collagen-binding physiologically active polypeptide according to any one of the above 1) to 9). A bacterium containing a gene encoding the collagen-binding bioactive polypeptide according to any one of the above 1) to 9). 15) Claim 1)
A transformant comprising a recombinant vector containing a gene encoding the collagen-binding bioactive polypeptide according to any one of to 9). A bacterium comprising a recombinant vector containing a gene encoding the collagen-binding physiologically active polypeptide according to any one of the above 1) to 9). Escherichia coli (Escherichia coli) containing a recombinant vector containing a gene encoding the collagen-binding physiologically active polypeptide according to any one of the above 1) to 9). 1)
A method for producing the collagen-binding physiologically active polypeptide according to any one of (9) to (9) using Esherichiacoli (Escherichia coli).

[0036]

DETAILED DESCRIPTION OF THE INVENTION Fibronectin (FN) is a cell-adhesive glycoprotein present on plasma, extracellular matrix and the surface of cultured cells.
It is known to bind to biological macromolecules such as heparin, fibrin and integrins and have biological effects such as cell adhesion, tissue construction and repair of tissue damage (Ruoslaht
i, Annual Review of Biochemistry, Vol. 57, No. 3
75-413 (1988)).

The collagen-binding bioactive polypeptide of the present invention is a polypeptide in which the above-mentioned FN-derived peptide is linked to a bioactive peptide, and has both collagen-binding activity and bioactivity. Shows the physiological activity after collagen binding. That is, it is a functional hybrid polypeptide in which a peptide derived from FN and a physiologically active peptide are linked by genetic engineering.

In the above-mentioned other studies, a hybrid polypeptide containing a collagen-binding peptide other than an FN-derived peptide and a physiologically active peptide was used.
It has been difficult to maintain both collagen binding activity and bioactivity. In particular, it is practically important for these hybrid polypeptides to exhibit physiological activity after binding to collagen, but this has not been sufficiently achieved conventionally.
The present inventors have also attempted to produce a hybrid polypeptide with FGF or EGF using a collagen-binding peptide of human von Willebrand factor, but have not obtained good results on collagen binding activity, I did not get enough and gave up.

By using an FN-derived collagen-binding polypeptide as the collagen-binding peptide as described above, both the collagen-binding activity and the physiological activity of the hybrid polypeptide can be maintained well, and the physiology after collagen-binding can be maintained. It was revealed that the biological activity of the active peptide was also maintained, and a novel functional hybrid polypeptide, a collagen-binding biologically active polypeptide, and its use were devised.

As a result, naturally, a physiologically active polypeptide having no collagen binding property naturally imparts a collagen binding property to a bioactive peptide having collagen binding property, depending on the FN-derived peptide. It became possible. Here, “collagen” is used to include collagen or heat-denatured collagen such as gelatin. Therefore, gelatin-binding property has the same meaning as collagen-binding property, and in this specification, these may be simply referred to as collagen-binding property or gelatin-binding property.

The collagen binding activity of the FN-derived peptide is strong and very stable even in the hybrid polypeptide. Then, after binding to collagen, it has become possible to complete a bioactive peptide-complexed collagen, which is a novel biomaterial that exhibits biological activity while maintaining the binding or being released slowly.

Further, the collagen-binding physiologically active polypeptide of the present invention has a
It has the property of being competitively inhibited by N, plasma, serum,
Exposure to blood, its addition or coexistence causes release from collagen, and it is retained by collagen in places where plasma, serum and blood are in short supply, that is, where humoral factors (cell growth factors) are starved. You have. Therefore, in the production of collagen-binding bioactive polypeptide,
The use of a collagen-binding peptide derived from FN as the collagen-binding peptide moiety is the key to the completion of the present invention.

At present, according to the recombinant DNA technology and the genetic engineering technology, in principle, it is basically possible to produce a hybrid gene (fusion gene) of any part of the FN-derived peptide gene with the bioactive peptide gene. It is possible. However, in order to produce a hybrid polypeptide while maintaining collagen binding properties and physiological activity, it is necessary to select an appropriate collagen binding peptide sequence. The selection of this sequence is also one of the present invention.

<FN-Derived Peptide (FNCBD)> That is, the FN-derived peptide constituting the present invention refers to FN
It is preferably a collagen-binding peptide derived from protease, which is obtained by protease degradation of FN, preferably limited degradation or spontaneous degradation.

Specifically, the above-mentioned FN-derived peptide is all homologous to the amino acid sequence constituting the polypeptide constituting the FN-derived collagen binding domain having binding activity to collagen and / or gelatin, or It is preferred that the sequence has one or several additions, or a sequence consisting of a partial substitution, deletion or addition thereof.

As the above-mentioned protease, trypsin, chymotrypsin, thermolysin, plasmin, thrombin, cathepsin D, pepsin and the like are preferably mentioned, and any one of these or a combination of two or more thereof is mentioned. The FN is preferably human FN, but may be FN of another animal species.

Accordingly, in the present specification, the collagen-binding domain of FN is specifically located between about 28 kDa and about 75 kDa from the amino terminus of FN, and is composed of trypsin, chymotrypsin, thermolysin, plasmin, thrombin, A collagen / gelatin binding polypeptide derived from FN that has been limitedly degraded by any of cathepsin D, cathepsin G, pepsin, subtilisin, chymase, leukocyte elastase or a combination of these proteases.

Specifically, collagen or gelatin-binding polypeptide of about 30 kDa obtained by limited digestion of trypsin or subtilisin, or limited digestion of chymotrypsin, limited digestion of cathepsin D and thrombin, limited digestion of leukocyte elastase A collagen or gelatin-binding polypeptide of about 39-45 kDa obtained by limited degradation of thermolysin or chymase or a polypeptide from Ala ²⁶⁰ to Trp ^{599 of} human FN obtained by limited degradation of plasmin and chymotrypsin. is there.

As another specific example of the collagen-binding FN peptide as described above, for example, an amino acid sequence constituting a polypeptide from Ala ²⁶⁰ to Trp ⁵⁹⁹ of human FN, or a sequence homologous or a part thereof Replacement,
Sequences that are deletions, insertions or adducts are included. As another specific example, as the human FN-derived peptide of the present invention, (1) Ala ²⁶⁰ of human FN obtained from human FN by limited or spontaneous degradation by a protease and having a binding activity to collagen / gelatin. Trp
An amino acid sequence up to ⁵⁹⁹ , a sequence homologous thereto, or a partial substitution, deletion, insertion or addition thereof, (2) a polypeptide from Ala ²⁶⁰ to Trp ^{599 of} human FN The amino acid sequence is homologous to the sequence, or is identical to a partially deleted or partially substituted, deleted, inserted or added product thereof, and has a carboxyl terminal derived from human FN. (3) Ala of human FN having a protease recognition sequence which is an amino acid
^A sequence containing the entire amino acid sequence constituting the polypeptide from ²⁶⁰ to Trp ⁵⁹⁹ , homologous to the sequence, or a sequence identical to a partial substitution, deletion, insertion or adduct thereof, ( 4) An amino acid sequence which is obtained from a polypeptide from Ala ²⁶⁰ to Trp ⁵⁹⁹ of human FN by limited or spontaneous degradation by a protease and constitutes a polypeptide having a binding activity to collagen / gelatin, or a sequence thereof. And sequences that are homologous to, or are partially substituted, deleted, inserted or added to.

The maintenance of gelatin binding in the hybrid polypeptide using the FN peptide as a polypeptide partner as described above can be confirmed, for example, by the fact that the polypeptide from Ala ²⁶⁰ to Trp ⁵⁹⁹ of human FN used in the present example is used. It can also be relatively easily confirmed by examining the gelatin binding activity of a polypeptide obtained by subjecting a hybrid polypeptide consisting of a physiologically active peptide to a limited degradation with the above protease. The degree of gelatin binding can be determined by maintaining binding in the presence of a high concentration of a salt such as 2 M NaCl, or by performing an experiment for inhibiting competition with plasma FN.

It should be noted that a slight difference from an amino acid sequence which is obtained by limited or spontaneous degradation by a protease and is homologous to an amino acid sequence constituting an FN-derived polypeptide having a binding activity to gelatin is a hybrid polypeptide. There are exceptions that do not diminish the collagen binding activity.

However, in most cases, even if it contains all or a part of the sequence of the collagen binding domain, it can be obtained only by a genetic engineering technique ignoring the protease degradation site or the site of spontaneous degradation. A hybrid polypeptide prepared by fusing an amino acid sequence derived from FN to a physiologically active peptide significantly reduces collagen binding activity. For example, human and FN Cys ²⁷⁷ to Ser ⁵⁷⁷ as disclosed in JP-A-62-89699.
Polypeptide consisting of amino acid sequence up to human FN
In a hybrid peptide obtained by fusing a polypeptide comprising an amino acid sequence from Thr ³⁷⁹ to Val ⁴⁴⁵ to a bioactive peptide by genetic engineering, collagen or gelatin binding activity is significantly impaired. Also, Japanese Patent Application Laid-Open
No. 89699 published by the inventors (Owens et al., EM
BOJ, Vol. 5, pp. 2825-2830 (1986)), a polypeptide from Thr ³¹⁴ to Met ⁴⁴⁶ of human FN,
Genetic engineering of human / FN polypeptide from Thr ³¹⁴ to Gly ⁵⁰⁷ , human / FN polypeptide from Thr ³⁷⁴ to Gly ⁵⁰⁷ , and human / FN polypeptide consisting of amino acid sequence from Thr ³⁷⁴ to Met ⁴⁴⁶ Even a hybrid peptide fused to a physiologically active peptide significantly impairs the gelatin binding activity.

Therefore, even when a hybrid collagen-binding bioactive peptide is prepared by a genetic engineering technique, the selection of the sequence of the collagen-binding peptide derived from FN requires the selection of the gelatin-binding peptide obtained by limited or spontaneous degradation by a protease. It is preferred to select and prepare a sequence homologous to the sex peptide.

The cleavage site by protease is, for example, C-terminal side of arginine and lysine in trypsin, C-terminal side of isoleucine, leucine, phenylalanine, tyrosine, tryptophan and threonine in chymotrypsin, and isoleucine, leucine, valine, phenylalanine and methionine in thermolysin. C-terminal side, C-terminal side of arginine and lysine in plasmin, between arginine and glycine in thrombin, C-terminal of lysine, tyrosine, phenylalanine and arginine in cathepsin D
Terminal side, C-terminal side of leucine, phenylalanine, tyrosine, methionine for pepsin, short side chain amino acids such as Gly, Ala or Val for leukocyte elastase
The C-terminal side of three consecutive amino acids, cathepsin G is the C-terminal side of leucine, tyrosine, and phenylalanine, and subtilisin is the C-terminal side of alanine.

However, in actual decomposition of FN, particularly in limited decomposition, there are portions that are easily cut and portions that are not easily cut depending on the higher-order structure. That is, the types of collagen / gelatin-binding peptides obtained by the limited degradation of proteases are very limited. Therefore, even when a partition of the collagen-binding peptide portion is selected by genetic engineering, a site that is easily cleaved by the protease is selected as the partition.

Specifically, a previous paper (Ruoslahti et al.,
J. Biol. Chem., 254, 6054-6059 (1979),
Balian et al., J Biol Chem., 254, pp. 1429-32,
(1979), Ruoslahti et al., J. Biol. Chem., Volume 254,
6054-6059 (1979), Hahn et al., Proc. Natl. Acad.
Sci., 76, 1160-1163 (1979), Gold et al., Pro.
c Natl Acad Sci US A., Vol. 76, pp. 4803-7, (197
9), Furie et al., J Biol Chem. 255, 4391-4,
(1980), Engvall et al., Coll. Relat. Res., Volume 1,
505-516 (1981), McDonald et al., J Biol Chem., No.
Vol. 256, pp. 5583-7 (1981), Vartiot et al., J Biol Che
m. 256, 471-7 (1981), De PetroG et al. Proc.
Natl Acad Sci US A. Vol. 78, pp. 4965-9 (1981), Ru
oslahti et al., J Biol Chem., 256, 7277-81 (19
81), Vartio et al. Eur J Biochem. 123, 223-33,
(1982), Petersen et al., Proc. Natl. Acad. Sci., No. 80.
137-141 (1983), Skorstengaard et al., Eur.
J. Biochem. 140: 235-243 (1984), Zardi
Eur. J. Biochem., 146, 571-579 (198
5), Skorstengaard et al., Eur. J. Biochem., No. 161.
Vol., Pp. 441-453 (1986), etc.) to know the sites of FN that are susceptible to cleavage by proteases and their gelatin binding properties, or by actually degrading FN with proteases to limit the amino terminus of peptides that bind to gelatin, There is a method of examining the site by examining the terminal or amino acid sequence.

Until now, many researchers have been studying the identification of collagen-binding peptides derived from FN, but there are conflicts in the reports of each researcher, and much remains to be known (Owens). Et al., EMBO J, Vol. 5,
2825-2830 (1986), Ingham et al., J. Biol. Chem.
264, pp. 16977-16980 (1989), Litvinovich
Et al., J. Mol. Biol, Vol. 217, 563-575 (199
1), Banyai et al., Eur. J. Biochem, 193, 801
806 (1990), Skorstengaard et al., FEBS letters,
343, 47-50 (1994)). According to the present inventors, it is determined that the discrepancies between these reports arise because the same consideration is given to the genetically produced peptide and the peptide obtained by protease treatment.

When producing an FN-derived peptide by genetic engineering, the separation of the sequence at an inappropriate site and the addition of a purified tag may cause the loss or reduction of the activity even in a region originally having collagen binding properties. It has become. Particularly in hybrid polypeptides, FN
Even if the peptide alone has collagen / gelatin binding properties, the binding properties are often not maintained. In other words, it causes the original higher-order structure to be distorted and no longer functions.

The collagen-binding bioactive peptide of the present invention is prepared by fusing a collagen-binding peptide of FN to which a sequence is divided at an appropriate site as described above and to which no purification tag is added, to the bioactive peptide. The collagen-binding physiologically active polypeptide of the present invention, as a recombinant hybrid peptide produced in Escherichia coli, almost all binds to gelatin due to the collagen / gelatin binding property of FN, and can be easily affinity-purified.
The bioactivity derived from the bioactive peptide is also maintained favorably.

The sequences of the collagen-binding peptides other than those derived from FN include collagenase and gelatinase which are matrix metalloproteases and von Willebrand factor in extracellular matrix.
Sequences derived from decorin, biglycan, fibromodulin, lumican, osteonectin, vitronectin, thrombospondin and the like have been reported. However, a hybrid polypeptide having a function equivalent to that of the present invention has not been reported, including an attempt to select a collagen-binding peptide derived from the above-mentioned bacterial collagenase and von Willebrand factor.

In the present invention, human-FN-derived peptides (for hybrids) are preferably exemplified by human-FN.
A polypeptide comprising an amino acid sequence of N from Cys ²⁷⁷ to Ser ⁵⁷⁷ (sequence described in JP-A-62-89699)
Examples of amino acid sequences that are suitable as FN-derived polypeptides of collagen-binding physiologically active polypeptides even if they do not include all of the above.

Specifically, Ala ^{260 of} human / FN obtained by limited degradation of plasmin, chymotrypsin and pepsin
To a polypeptide composed of the amino acid sequence from Leu ³⁷⁶ to plasmin, a polypeptide composed of the amino acid sequence from Ala ²⁶⁰ to Leu ⁴⁸³ of human / FN obtained by limited degradation of chymotrypsin and pepsin, and a limited degradation of trypsin Arg ⁴⁸⁴ from Ala ^{260 of} human and FN obtained
Polypeptide consisting of the amino acid sequence of plasmin, chymotrypsin and pepsin obtained by the limited degradation of human FN polypeptide consisting of the amino acid sequence from Ala ³⁷⁷ to Leu ⁴⁸³ , limitation of plasmin, chymotrypsin and pepsin Val of human and FN obtained by decomposition
A polypeptide consisting of the amino acid sequence from ³⁷⁷ to Trp ⁵⁹⁹ , Leu ⁴⁸³ to Trp ⁵⁹⁹ of human FN obtained by limited degradation of plasmin, chymotrypsin and pepsin
A polypeptide composed of the amino acid sequence up to, human F obtained by limited digestion of trypsin and chymotrypsin
1 is the amino acid sequence of the polypeptide from N Arg ⁴⁸⁴ to Trp ⁵⁹⁹ .

A polypeptide sequence containing all of the polypeptide sequence consisting of the amino acid sequence from Cys ²⁷⁷ to Ser ⁵⁷⁷ of human FN and suitable as a FN-derived polypeptide of a collagen-binding physiologically active polypeptide And specifically, Ala of human / FN obtained by limited degradation of plasmin and chymotrypsin.
Val from human FN obtained by limited degradation of polypeptides from ²⁶⁰ to Trp ⁵⁹⁹ , subtilisin and chymotrypsin
Amino acid sequences of polypeptides from ²⁶² to Trp ⁵⁹⁹ .

Even if it contains all of the polypeptide sequence consisting of the amino acid sequence from Cys ²⁷⁷ to Ser ⁵⁷⁷ of human FN, it is not suitable as a FN-derived polypeptide of collagen-binding physiologically active polypeptide. There are many examples of such polypeptides, and there are many examples. For example, the amino acid sequence of Asn ²²⁰ to Ser ⁵⁷⁷ of human FN The amino acid sequence of Arg ²²¹ to Ser ⁵⁷⁷ of human FN Human Amino acid sequence from Gly ²²² to Ser ⁵⁷⁷ of FN Human Amino acid sequence from Asn ²²³ to Ser ⁵⁷⁷ of FN Human Amino acid sequence from Leu ²²⁴ to Ser ⁵⁷⁷ of FN Human Amino acid sequence from Leu ²²⁵ to Ser ^{577 of} FN amino acid sequence human · FN from sequence human · FN of Gln ²²⁶ of the amino acid sequence human · FN of Ile ²²⁸ of the amino acid sequence human · FN of Cys ²²⁷ to Ser ⁵⁷⁷ to Ser ⁵⁷⁷ to Ser ⁵⁷⁷ Amino acid sequence from Cys ²⁷⁷ of the amino acid sequence human FN of Cys ²⁷⁷ to Cys ¹²⁰¹ of the amino acid sequence human FN of Cys ²⁷⁷ of the amino acid sequence human FN of Cys ²⁷⁷ to Thr ¹²⁰² to Phe ¹²⁰³ to Asp ¹²⁰⁴ human Amino acid sequence from Cys ²⁷⁷ to Asn ¹²⁰⁵ of FN Amino acid sequence from Cys ²⁷⁷ to Leu ¹²⁰⁶ of human / FN Amino acid sequence from Cys ²⁷⁷ to Ser ¹²⁰⁷ of human / FN Amino acid sequence from Cys ²⁷⁷ to Pro ¹²⁰⁸ of human / FN A polypeptide composed of the amino acid sequence of human / FN from Cys ²⁷⁷ to Gly ¹²⁰⁹ and the like can be mentioned.

From the above, Cys ²⁷⁷ of Ser.
The selection of the amino acid sequence of the FN-derived peptide of the collagen-binding physiologically active peptide of the present invention may or may not include all of the amino acid sequence of the polypeptide up to ⁵⁷⁷ (the sequence described in JP-A-62-89699). It doesn't matter. Since the amino acid sequence of Thr ³⁷⁹ to Val ⁴⁴⁵ is contained in both cases where it is appropriate or inappropriate as an FN-derived peptide, sufficient conditions for the collagen-binding physiologically active peptide of the present invention as an FN-derived peptide can be obtained. Absent. In Japanese Patent Application Laid-Open No. 62-89699, Thr ³⁷⁹ to Val ⁴⁴⁵ are described as a continuous portion having a collagen binding ability.
ters, Vol. 343, pp. 47-50 (1994)) from Thr ³⁷⁹
It has been reported that a polypeptide comprising the amino acid sequence from The ³⁷⁴ to Ala ⁴⁷⁹ , including Val ⁴⁴⁵ , alone had no collagen binding ability.

<Bioactive peptide> The term "bioactive peptide" as used herein is a general term for peptides, polypeptides and proteins having various physiological activities, and includes, for example, cytokines and cell growth factors. For example, fibroblast growth factor (FGF) family, transforming growth factor β (TGF-β) family, epidermal growth factor (EGF) family, platelet-derived growth factor (P
Cell growth factors such as DGF), insulin-like growth factor (IGF), nerve growth factor (NGF) family, vascular endothelial cell growth factor (VEGF), hepatocyte growth factor (HGF) and bone morphogenetic factors (BMPs) Cell growth factors including cell differentiation factors such as interferon (IF
N), interleukins (ILs), colony stimulating factors (CSF), erythropoietin, tumor necrosis factor (T
Examples include cytokines including NF), various hormones such as insulin and parathyroid hormone (PTH), and enzymes such as proteases such as matrix metalloproteases (MMP). However, usually, maltose binding protein (MBP), glutathione-S-
Transferase (GST), histidine tag, β-galactosidase and the like are not included in the bioactive peptide herein.

The term “bioactivity” or “activity of a bioactive peptide” as used herein means a part or all of the activity derived from the above-mentioned bioactive peptide. Differentiation activity,
It is a migratory activity or substance production promotion, etc., and is not merely binding to cells or receptors. The activity derived from FN is not included in the bioactivity herein.

The function of the fusion partner or the purification tag as described above, for example, the binding activity to a specific substance, such as the affinity of thioredoxin to a metal chelate, the binding of protein A to IgG, the chloram Binding of phenicol acetyl transferase to chloramphenicol, binding of lac repressor to lac operator, binding of galactose binding protein to galactose, amylose binding in MBP, glutathione binding in GST, histidine The tag does not include the nickel-binding property and any or all of the enzyme activity of β-galactosidase in the present specification.

The binding or enzymatic activity of these fusion partners and purified tags has been demonstrated to maintain their functions with various hybrid peptides and to impair the activity of polypeptides fused with these fusion partners and purified tags. It is a well-known fact that it is difficult to break, and it can be said that it is a special case.

<Hybrid polypeptide> In the present invention, the fibronectin collagen-binding domain (F
NCBD) and a bioactive peptide are linked by a genetic engineering technique, and the bioactive peptide is FNCB.
It is desirable that D be linked to the carboxyl terminal side of D. The collagen-binding bioactive polypeptide of the present invention is water-soluble. Also bacteria, preferably Esherichia c
Oli can be produced industrially. Therefore, it is desirable that the bioactive peptide be linked to the carboxyl terminus of the fibronectin-derived peptide by a genetic engineering technique and produced in bacteria, preferably in Escherichia coli. It can also be produced in yeast, insect cells, and animal cells.

For example, a bioactive peptide is linked to the carboxyl terminal side of the collagen binding domain of FN,
It is possible to insert an arbitrary amino acid sequence that can be cleaved by a protease (for example, existing in a human body) into the amino terminal side of the bioactive peptide, and to release the bioactive peptide without adding an extra sequence. . More specifically, since many proteases cleave the carboxyl terminus of the recognition sequence, if a protease recognition sequence is added to the amino terminus of the bioactive peptide, an extra amino acid sequence will be added to the amino terminus of the bioactive peptide after cleavage by the protease. It does not remain. In addition to newly inserting the protease recognition sequence, the C-terminal side of the collagen binding domain of FN may be a protease recognition sequence and a cleavage site. Therefore, if a physiologically active peptide is directly linked to the carboxyl terminus, it is possible to exclude insertion of an artificial sequence at all. After cleavage at this site, no extra amino acid sequence remains in both the collagen-binding domain of FN and the physiologically active peptide. If the mode of exerting bioactivity on cells by binding to the receptor from the solid phase of matrixrine and juxtacline activity is possible, it may be possible to bind to the receptor and show bioactivity even with the hybrid polypeptide as it is . On the other hand, if the bioactive peptide is cleaved from the collagen-binding domain of FN and needs to be released into the liquid phase, the above method is important.

The collagen-binding bioactive polypeptide of the present invention in which FNCBD and the bioactive peptide are linked as described above maintains both the binding activity to collagen and the bioactivity. Can be stably retained in a collagen matrix, and physiological activity can be continuously exhibited.

In the above, an amino acid or a peptide may be inserted as a spacer at the connecting site between the physiologically active peptide and the peptide derived from the fibronectin.
In this case, it is preferable that the spacer and any one of the spacer and a sequence adjacent thereto include a protease recognition sequence. The protease recognition sequence is preferably an enterokinase recognition sequence.

The enterokinase recognition sequence can be used as a spacer to adjust the distance between FNCBD and bFGF or EGF or to enter FKBD into bFGF by enterokinase.
Alternatively, it plays a role in releasing EGF. Enterokinase is a protease present in the duodenal mucosa and pancreas of higher animals. Therefore, the enterokinase recognition sequence is recognized and cleaved by enterokinase in the duodenal mucosa and pancreas, and bFGF or EGF is released. Enterokinase is an enzyme that recognizes DDDDK, a very specific sequence, and cleaves the C-terminal side of K (Lys), and is necessary for studying the mechanism of the physiological activity of collagen-binding bioactive peptide. Is an array. In the future, collagen-binding physiologically active peptide is indispensable for industrial use.

The collagen-binding activity or collagen-binding activity of the hybrid polypeptide of the present invention is a binding to collagen dependent on an FN-derived peptide,
It is not binding to collagen dependent on bioactive peptides. Whether the collagen binding activity is derived from FN or a bioactive peptide can be confirmed by a competitive inhibition experiment using natural FN or a bioactive peptide.

[0076] Collagen or a polypeptide derived from collagen as referred to in the present specification is natural collagen, immature collagen, atelocollagen, gelatin or a polypeptide constituting them.

As one application of the hybrid polypeptide of the present invention, a local maintenance agent, sustained release agent or bioactivity imparting agent for a physiologically active polypeptide containing the collagen-binding physiologically active polypeptide is provided. In addition, a bioactive peptide is sustainedly released by degradation of collagen or by cleavage by a protease at or near the site where the bioactive peptide and the FN-derived peptide are linked, thereby providing a biomaterial with controlled bioactivity. It is possible to provide a method for imparting the biological activity of proliferation, differentiation, regeneration or promotion of substance production to cells, tissues and organs using the above biomaterial.

Therefore, for example, in order to exert the effect of healing wounds after trauma or surgery or to promote the healing of chronic ulcers and intractable ulcers continuously and maximally, the sustained release or local release of cell growth factors such as bFGF and EGF A collagen-binding cell growth factor and a cell growth factor-complexed collagen matrix for retention are provided. Biological tissue is composed of collagen in a large proportion, and collagen is present at all sites.If the bioactive polypeptide has collagen-binding properties, it will bind to collagen at or near the site to which it is administered. Maintain high local concentrations, increase effectiveness. At the same time, side effects caused by diffusion to other parts are prevented.

The present invention also provides a method for cell proliferation using the above collagen-binding physiologically active polypeptide (hybrid polypeptide). That is, the hybrid polypeptide in which the physiologically active peptide of the present invention is a cell growth factor can be immobilized on a collagen / gelatin matrix by utilizing its collagen / gelatin binding property, and the cell growth factor flows out of the perfused culture solution. Therefore, cell growth can be easily performed.

The present invention is not limited to the purpose of wound healing using bFGF and EGF, but is applicable to many peptides having bioactivity. That is, according to the present invention, the physiological activity of various physiologically active peptides is maintained, and a collagen-binding biologically active polypeptide aimed at sustained release or local retention of the physiologically active peptide by imparting a binding activity to collagen is provided. Provided.

A gene encoding the above collagen-binding physiologically active polypeptide, a recombinant vector containing the gene, a transformant containing the gene, or a bacterium containing the gene, preferably Escherichia
a coli is also provided. Alternatively, a transformant containing a recombinant vector containing a gene encoding a collagen-binding physiologically active polypeptide, and a bacterium, preferably Escherichia coli, containing the vector are also provided.
Further, a vector for gene therapy is provided by incorporating the gene encoding the collagen-binding bioactive polypeptide into a retrovirus, an adenovirus, a virus vector such as an adeno-associated vector, or a vector using a liposome method or the like. By introducing a vector into a cell, a cell medicine containing the polypeptide or the gene is provided.

A complex of the polypeptide with collagen is provided with a physiologically active peptide-complexed collagen matrix, which is functionally modified by its activity, and is useful as a novel tissue regeneration biomaterial used in the medical field. That is, the present invention relates to artificial tissues (blood vessels, nerves, bones, etc.), artificial organs having three-dimensional structures (ears, nose, fingers, skin, etc.) and artificial organs (intestinal tract such as duodenum, stomach, heart,
A biomaterial comprising a bioactive peptide-complexed collagen having both a scaffold for constructing a liver, a pancreas, a kidney, etc.) and a bioactivity. FIG. 2 shows a conceptual diagram of the collagen matrix complexed with the collagen bioactive peptide.

Hereinafter, embodiments of the present invention will be described in more detail. The primary structures of human FN cDNA and protein are respectively described in EMBL DATA BANK.
And The EMBO Journal, Volume 4, Issue 7, pages 1755-1759
(1985).

First, a sequence corresponding to the amino acid sequence of the collagen / gelatin binding domain obtained by subjecting human FN to limited degradation with proteases (plasmin and chymotrypsin) was cloned. M extracted from human cells
A reverse transcription reaction is performed using RNA, and a PCR method (Polymerase Chain Reaction: Saiki
Et al., Science, Vol. 230, pp. 1350-1354 (1
985)), the cDNA portion corresponding to the collagen binding domain (FNCBD) of human FN is amplified.
At this time, 5 ′ is included in the sense primer used for PCR.
A restriction enzyme recognition sequence and a start codon sequence are added to the end, and a restriction enzyme recognition sequence and a termination codon are added to the 5 ′ end of the antisense primer.

Then, the cDNA fragment of FNCBD is inserted into the cloning vector pBlueScript SK, and the plasmid pBS (FNCBD) is constructed. After confirming the nucleotide sequence, this cDNA fragment is cut out and incorporated into an expression vector pTYB1 to construct a plasmid pTYB1 (FNCBD).
pTYB1 (FNCBD) is a human FN Ala ²⁶⁰ -Trp ⁵⁹⁹
(340 amino acid residues).
A collagen-binding polypeptide is prepared by introduction into E. coli.

The FNCBD cDN required in the present invention
As A, cDN derived from plasmid pBS (FNCBD)
The A fragment is used, but the initiation codon sequence is added to the 5 'end of the cDNA fragment by the design of the PCR primer, and a cloning site such as XhoI just before the stop codon added to the C terminal of the FNCBD translation region.
A recognition sequence has been introduced. Thereby, FNCBD
Can be linked to the cDNA of the physiologically active peptide.

The polypeptide of the present invention comprises c
It is prepared by ligating DNA and cDNA of a physiologically active peptide and expressing them by genetic engineering. The functional polypeptide according to the present invention is obtained, for example, by converting human FN represented by SEQ ID NO: 1 into a protease (plasmin and chymotrypsin).
An artificial function in which a polypeptide of 340 amino acid residues corresponding to Ala ²⁶⁰ -Trp ⁵⁹⁹ obtained by limited digestion with human bFGF or EGF represented by SEQ ID NO: 2 or 3 in the Sequence Listing, respectively. Sex polypeptide. That is, the polypeptide is prepared by genetic engineering by linking bFGF or EGF cDNA to FNCBD cDNA.

Amino acid number 1 in SEQ ID NO: 1 corresponds to Met corresponding to a start codon for expressing human FNCBD by genetic engineering, and amino acid numbers 2 to 341 correspond to human FN.
The CBD amino acid sequence, amino acids 342-343, contains XhoI for linking to the bioactive peptide cDNA.
These are the amino acids Leu and Glu derived from the recognition sequence. However, in the hybrid polypeptide, the XhoI recognition sequence and Sal
Glu is removed by ligation of the I recognition sequence.

Amino acid number 1 in SEQ ID NO: 2 is FN
Asp derived from SalI recognition sequence for ligation with CBD cDNA, amino acids 1 to 5 are recognition sequences of enterokinase (Asp-Asp-Asp-Asp-Lys; DDDDK), amino acids 6 to 159 are human bFGF Amino acid sequence.

Amino acid number 1 in SEQ ID NO: 3 is FN
Asp derived from the SalI recognition sequence for linking to the CBD cDNA, amino acids 1 to 5 are the enterokinase recognition sequence (DDDDK), and amino acids 6 to 58 are the amino acid sequence of human EGF.

The superscript attached to the amino acid of human FN indicates the number of amino acid residues counted from the N-terminal of mature human FN in the EMBL DATABANK. Also, human FN represented by SEQ ID NO 1 Ala ²⁶⁰ -
The polypeptide of 340 amino acid residues corresponding to Trp ⁵⁹⁹ differs from the amino acid sequence in EMBL DATA BANK by 2 amino acid residues and is not an artificial mutation.

The primary structure of human bFGF cDNA and protein is described in Kurokawa et al., (FEBS Letter, Vol. 213,
No. 1, pages 189-194 (1987)). The primary structure of human EGF cDNA and protein is described in Bell et al. (Nucleic Acids Research, 14th edition).
Vol. 21, No. 8, pp. 8427-8446 (1986)).

In the present invention, human bFGF and Eb were prepared by PCR from cDNA prepared using human mRNA.
The GF cDNA is amplified. For each PCR, a sense primer having a restriction enzyme recognition sequence and a nucleotide sequence encoding a protease recognition sequence added to the 5 ′ end and an antisense primer having a restriction enzyme recognition sequence added to the 5 ′ end were used. Next, these cDNA fragments were converted into pBl
Insert into uescripSK, pBS (FGF) vector and pBS
An (EGF) vector is created.

The above-mentioned cDNA of human FNCBD is ligated to the bFGF or EGF 5′-terminal restriction enzyme recognition sequence of plasmid pBS (FGF) or pBS (EGF), and human bFGF or EGF is added to the C-terminus of human FNCBD. Plasmid pBS (FN
CBD-FGF) or pBS (FNCBD-EGF) is obtained. The hybrid gene fragment excised from these plasmids is inserted into an expression vector pTYB1, and a recombinant plasmid pTYB1 (FNCBD-FG) expressing the polypeptide represented by SEQ ID NO: 4 or SEQ ID NO: 5 in the Sequence Listing is obtained.
F) or pTYB1 (FNCBD-EGF) is obtained.

FNCBD cDNA and bFGF or E
A base sequence derived from a PCR primer is inserted into a GF cDNA linking site. By expressing an enterokinase recognition sequence or the like as a spacer peptide, the intermolecular distance between bFGF or EGF to FNCBD and / or protease Insertion of a recognition sequence is possible. The presence or absence of the protease recognition sequence in the spacer, the type, the peptide sequence, and the length are selected depending on the purpose.

Sequence Listing SEQ ID NO: 4 is a hybrid polypeptide of human FNCBD and human bFGF, amino acid No. 1 is Met corresponding to the start codon, amino acid Nos.
341 is the amino acid sequence of human FNCBD, and amino acids 342 to 343 are the cDNA of human FNCBD and bFGF
Leu and Asp encoded by the nucleotide sequence generated by ligation with the cDNA (ligation of the XhoI recognition sequence and the SalI recognition sequence), amino acids 343 to 347 are the enterokinase recognition sequence (DDDDK),
５０501 is the amino acid sequence of human bFGF.

Sequence Listing SEQ ID NO: 5 is a hybrid polypeptide of human FNCBD and human EGF, amino acid No. 1 is Met corresponding to the start codon, amino acids Nos. 2-34
1 is the amino acid sequence of human FNCBD, amino acid No. 34
2-343 are human FNCBD cDNA and EGF cD
Leu and Asp encoded by the nucleotide sequence generated by ligation with NA (ligation of XhoI and SalI sites)
Amino acids 343-347 are the enterokinase recognition sequence (DDDDK), amino acids 348-400 are human E
2 is the amino acid sequence of GF.

The plasmid pTYB1 (F
NCBD), pTYB1 (FNCBD-FGF) and pTYB1 (F
NCBD-EGF) is introduced into E. coli and cultured under appropriate conditions, whereby the target polypeptide is accumulated in E. coli. Immunoblotting is used to confirm expression. That is, whole cell proteins of the recombinant Escherichia coli were separated by SDS-polyacrylamide gel electrophoresis, and after transfer of nitrocellulose membrane, FNCBD, bFGF
And a band of the target polypeptide is detected by a monoclonal antibody that recognizes EGF and EGF, respectively.

The polypeptide of the present invention is prepared, for example, as follows. The recombinant Escherichia coli with the introduced plasmid
The cells are cultured in a medium such as SB broth, and IPTG (isopropyl-β-D-galactoside) is added. Thereby, the expression of the introduced gene is induced, and the cells are recovered after further culturing. The collected cells are disrupted by sonication. Then, the target polypeptide is obtained as an inclusion body in the precipitate recovered by centrifugation from the cell lysate, and denatured and solubilized with 8M urea.

Next, dialysis is performed while the urea concentration is gradually reduced, whereby the target protein is reactivated (refolding treatment). 40 kDa prepared as above,
The 57 kDa and 46 kDa polypeptides are each FNCBD
(FIG. 1A), a hybrid polypeptide of FNCBD and bFGF (FNCBD-FGF, FIG. 1B) and F
A hybrid polypeptide of NCBD and EGF (FN
CBD-EGF, FIG. 1C), wherein B and C are specific examples of a collagen-binding bioactive polypeptide.

The collagen binding activity of the polypeptide prepared as described above is examined by two methods. One is a batch method of gelatin (Gelatin Sepharose 4B)
, Amersham Pharmacia Biotech). After adding the above-mentioned polypeptide and a turbid solution of gelatin sepharose 4B to a 1.5 ml microtube, the mixture is mixed with a rotator at 4 ° C. for 1 hour, and after centrifugation, the supernatant is recovered. The precipitated fraction is 1M sodium chloride solution
After washing twice, the supernatant is recovered. 1M with the same operation
Elution of the polypeptide bound to gelatin is attempted sequentially with urea, 2M urea, and 4M urea. Finally, the supernatant is recovered by boiling in sodium dodecyl sulfate (SDS) buffer. In SDS-polyacrylamide electrophoresis (SDS-PAGE) of the collected supernatant, binding of the polypeptide to gelatin and elution from gelatin are examined based on the presence or absence of a band.

Another approach is to use FNCBD, FNCBD-FGF and FNCB for the collagen in different states, ie gelatin, natural collagen and atelocollagen.
The binding activity of D-EGF is examined. First, gelatin,
After native collagen and atelocollagen were each coated on a 96-well plate, they were blocked with serum albumin, FNCBD, FNCBD-FGF and FNC.
After dispensing each BD-EGF, it is incubated at 37 ° C. for about 1 hour. After washing, addition of an anti-FNCBD monoclonal antibody, washing, a labeled secondary antibody reaction, and washing, the collagen binding activity is determined by measuring the absorbance of the color developed by the addition of the substrate.

The cell proliferation activity of each collagen-binding bioactive polypeptide is directly correlated with the number of viable cells by measuring the mitochondrial respiratory chain dehydrogenase activity.
(Roehm et al., Journal Immunological Methods, 142
Volume, 257-265 (1991)) or the WST-1 method (Liu et al.,
Nature Medicine, Vol. 1, 267-271 (1995)). That is, mouse BALB / c3T3 cells or human fibroblasts are cultured for 4 days in a medium to which each of the polypeptides has been added, then XTT or WST-1 reagent is added, and the cells are further cultured for 3-6 hours. Thereafter, the cell growth activity of the polypeptide can be measured by measuring the absorbance of the medium at a specific wavelength.

In the same manner as described above, the cell proliferation activity of a collagen matrix in which a collagen-binding physiologically active polypeptide is conjugated is examined by the XTT method or the WST-1 method. Different amounts of the polypeptide solution are added to a collagen-coated cell incubator and left at 37 ° C. After one hour, the solution is removed, and after washing with a phosphate buffer, a collagen matrix in which each physiologically active peptide is complexed is formed. BALB / c3T3 cells, NRK49F rat fibroblasts or human fibroblasts are cultured on this matrix for 4 days. Further, XTT or WST-1 reagent is added, and the cells are cultured for 3 to 6 hours. Thereafter, by measuring the absorbance at a specific wavelength, the cell growth activity of the bioactive peptide-complexed collagen matrix can be measured.

[0105]

EXAMPLES Hereinafter, the feasibility of the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. (Example 1) Preparation of a hybrid polypeptide of human FNCBD and human bFGF and a hybrid polypeptide of human FNCBD and EGF (a) Cloning of cDNA encoding human FNCBD Using mRNA extracted from human kidney cells as a template Using the primer (2), the cDNA was reverse transcribed, and a pair of primers (1) and (2) was used to PCR amplify the human FNCBD cDNA corresponding to the sequence obtained by the limited digestion with plasmin and chymotrypsin. (RT-PCR).

The nucleotide numbers 1 to 2 of the primer (1) represented by SEQ ID NO: 6 are adjacent additional sequences prepared for KpnI digestion after PCR, and the nucleotide numbers 3 to 8 are KpnI recognition sequences for cloning, Nucleotide Nos. 7 to 12 are NcoI recognition sequence, Nucleotide Nos. 13 to 14 are sequences matching expression frame, Nucleotide Nos. 15 to 20 are NdeI recognition sequence, Nucleotide No. 18
20 to 20 are initiation codon sequences for expressing human FNCBD, and base numbers 21 to 49 are base sequences of human FNCBD.

The base numbers 1-2 of the primer (2) represented by SEQ ID NO: 7 in the sequence listing correspond to BamHI after PCR.
Flanking additional sequences prepared for digestion, base numbers 3 to 8 are BamHI recognition sequences for cloning, base numbers 9 to 11 are stop codon antisense sequences, and base numbers 12 to 17 are bF
XhoI recognition sequence for linking to a cDNA of a physiologically active peptide such as GF or EGF, base numbers 18 to 46
Is the antisense sequence of the human FNCBD cDNA.

RT-PCR was performed using RNA LAPCR Kit (AMV) Ve
r.1.1 (Takara Shuzo) first, total RNA 0.8μ
g in a 20 μl reaction volume at 60 ° C. for 20 minutes,
It was further heated at 99 ° C. for 5 minutes. A PCR reaction using the obtained cDNA as a template was carried out in a 100 μl reaction volume at 94 ° C. for 1 minute and then at 94 ° C. for 30 seconds → 6
A temperature cycle of 1 minute at 3 degrees → 2 minutes at 72 degrees was repeated 12 times. A 1/10 volume of the reaction solution was analyzed by agarose gel electrophoresis. As a result, a DNA fragment of about 1 kbp was observed.
This was the size corresponding to the cDNA of human FNCBD.

The amplified cDNA fragment was digested with KpnI and BamHI, and ligated to the cloning vector pBluescriptSK digested with KpnI and BamHI at 25 ° C. for 3 minutes (using Takara Shuzo Ligation Kit Ver. 2).

As a result of the nucleotide sequence analysis, a plasmid into which the cDNA represented by SEQ ID NO: 8 was incorporated was obtained and named pBS (FNCBD). Nucleotide numbers 1 to 6 in the sequence listing SEQ ID NO: 8 are KpnI recognition sequences for cloning, and nucleotide numbers 5 to 10 are NcoI recognition sequences, nucleotide numbers.
11-12 is a frame alignment sequence, base numbers 13-18 are
The NdeI recognition sequence, base numbers 16 to 18 are human FNCBD
Start codon sequence for expressing, base numbers 19 to 1038
Is the nucleotide sequence of human FNCBD cDNA, nucleotide number 10
39-1044 are XhoI recognition sequences for linking to a cDNA of a bioactive peptide such as bFGF or EGF;
Nucleotide numbers 1045 to 1047 are stop codons, base numbers 1048 to 10
53 is a BamHI recognition sequence for cloning. The FNCBD cDNA sequence was different from the sequence registered in EMBL DATA BANK by 5 bases, but it was confirmed that this difference was not caused by point mutation by PCR.

(B) cDNA encoding human bFGF
Using mRNA extracted from human kidney cells as a template, cDNA was reverse transcribed using primer (4) as a template, and cDNA of human bFGF was amplified by PCR using a pair of primers (3) and (4). . Base numbers 1-2 of the primer (3) represented by SEQ ID NO: 9
Is a flanking additional sequence for SalI digestion after PCR, and nucleotides 3 to 8 are for cloning and FNCBD cDNA.
A base sequence encoding a recognition sequence (DDDDK) for enterokinase,
Nucleotide numbers 21 to 40 are the nucleotide sequence of the cDNA of human bFGF.

The base No. 1 of the primer (4) represented by SEQ ID No. 10 in the Sequence Listing is an adjacent additional sequence prepared for EcoRI digestion after PCR, the base Nos. 2 to 7 are an EcoRI recognition sequence for cloning, Nos. 8 to 10 are the antisense sequence of the stop codon, and base numbers 11 to 31 are human bF
Antisense sequence of GF cDNA.

RT-PCR was performed using RNA LAPCR Kit (AMV) Ver.
1.1 Using (Takara Shuzo), first, the total R of the template
0.8 μg of NA was reverse-transcribed in a reaction solution volume of 20 μl at 60 ° C. for 20 minutes, and further heated at 99 ° C. for 5 minutes. The PCR reaction was performed at 94 ° C. in a 100 μl reaction solution containing cDNA.
Then, a temperature cycle of 94 ° C. for 30 seconds → 65 ° C. for 4 minutes was performed 30 times. As a result of analyzing a 1/10 amount of the reaction solution by agarose gel electrophoresis, a DNA fragment of about 450 bp was observed. This was the size corresponding to human bFGF.

This cDNA fragment was prepared using SalI and EcoR.
PBlues digested with SalI and EcoRI after digestion with I
I ligated to criptSK. Then, a plasmid into which the cDNA represented by SEQ ID NO: 11 was incorporated was obtained and named pBS (FGF). Nucleotide numbers 1 to 6 of SEQ ID NO: 11 in the Sequence Listing are
SalI recognition sequence for ligation to DNA, base numbers 4 to 18 are base sequences encoding enterokinase recognition sequence (DDDDK), base numbers 19 to 480 are base sequences of human bFGF cDNA, base numbers 481 to 483 are termination. Codon, base number 48
4-489 are EcoRI recognition sequences for cloning. The nucleotide sequence of the obtained human bFGF was EMBL
Sequence and 1 registered in the data bank (EMBL DATABANK)
Although the bases were different, this difference was due to a point mutation by PCR. However, since it was a silent point mutation with no amino acid change, it was used as it was.

(C) Cloning of cDNA encoding human EGF Reverse transcription of cDNA extracted from cells of human kidney into cDNA using primer (6) as a template, and a set of primers (5) and (6) Was used to PCR amplify the cDNA of human EGF. Base numbers 1-2 of the primer (5) represented by SEQ ID NO: 12
Is the adjacent additional sequence prepared for SalI digestion after PCR, and base numbers 3 to 8 are for cloning and FNCBD cDNA.
A base sequence encoding a recognition sequence (DDDDK) for enterokinase,
Nucleotide numbers 21 to 44 are the nucleotide sequences of human EGF cDNA.

Further, base number 1 of the primer (6) represented by SEQ ID NO: 13 in the sequence listing is an adjacent additional sequence prepared for EcoRI digestion after PCR, base numbers 2 to 7 are an EcoRI recognition sequence for cloning, and base numbers. 8 to 10 are the antisense sequence of the stop codon, and base numbers 11 to 30 are the antisense sequence of the human EGF cDNA.

RT-PCR was performed using TaKaRa RNA LA PCR K
Using it (AMV) Ver.1.1 (Takara Shuzo), 1.0 μg of total template RNA was first used in a reaction volume of 20 μl.
The reverse transcription reaction was performed at 0 ° C. for 30 minutes, and the mixture was heated at 99 ° C. for 5 minutes. The PCR reaction was carried out at 94 ° C. for 1 minute in a reaction solution volume of 100 μl, and then subjected to 35 temperature cycles of 94 ° C. for 30 seconds → 65 ° C. for 45 seconds 35 times.

[0118] A 1/10 amount of the reaction solution was analyzed by agarose gel electrophoresis, and as a result, a DNA fragment of about 150 bp was observed. This was the size corresponding to human EGF. After digesting this DNA fragment with SalI and EcoRI,
Ligation was performed on pBluescriptSK digested with II and EcoRI. Then, a plasmid into which the cDNA represented by SEQ ID NO: 14 was incorporated was obtained, and pBS
(EGF).

Nucleotide numbers 1 to 6 in SEQ ID NO: 14 are used for cloning and for linking to the FNCBD cDNA.
lI recognition sequence, base numbers 4 to 18 are base sequences encoding enterokinase recognition sequence (DDDDK), base numbers 19 to 17
7 is the base sequence of human EGF cDNA, base numbers 178 to
180 is a termination codon sequence, and base numbers 181 to 186 are EcoRI recognition sequences for cloning. The nucleotide sequence of human EGF obtained was obtained from EMBL Data Bank (EMBL DAT
A BANK).

(D) Preparation of human FNCBD expression vector Plasmid pBS (FNCBD) obtained in (a) above
With NdeI and NotI (NotI recognition sequence is pBlues
criptSK at the multiple cloning site). The inserted FNCBD cDNA fragment was cut out, and the expression vector pTYB1 (New England BioL) was used.
ab)) ligation to NdeI-NotI site. The constructed plasmid was named pTYB (FNCBD).

(E) Preparation of hybrid polypeptide expression vector of FNCBD and bFGF Plasmid pBS (FNCBD) obtained in (a) above
Was digested with KpnI and XhoI. EMBL DATA
Although no XhoI recognition sequence exists in the sequence registered in BANK, FNCBD obtained by RT-PCR from kidney RNA was used.
Since the XhoI recognition sequence was present in the cDNA sequence of the above, partial digestion was carried out.

The FNCBD inserted by this process
Was excised and ligated to the KpnI-SalI site of the plasmid pBS (FGF) obtained in (b) above. The thus constructed plasmid incorporates the DNA represented by SEQ ID NO: 15 in the Sequence Listing, and the plasmid is called pBS (FNCBD-
FGF).

Nucleotide numbers 1 to 6 in SEQ ID NO: 15 are KpnI recognition sequences for cloning, and nucleotide numbers 5 to 10 are Nc
oI recognition sequence, base numbers 11 to 12 are frame alignment sequences, base numbers 13 to 18 are NdeI recognition sequences, base numbers 16 to
18 is the start codon sequence, base numbers 19 to 1038 are human FNCB
D cDNA sequence, base numbers 1039 to 1044 are base sequences generated by ligation of the XhoI recognition sequence and SalI recognition sequence, base numbers 1042 to 1056 are base sequences encoding the enterokinase recognition sequence (DDDDK), base number 1057
~ 1518 is the cDNA sequence of human bFGF, base numbers 1519 ~
1521 is a stop codon, and base numbers 1522 to 1527 are EcoRI recognition sequences for cloning.

The DNA fragment of the hybrid gene inserted into pBS (FNCBD-FGF) was converted to NdeI and Ec
The fragment was excised with oRI and the fragment
Ligation was performed at the NdeI-EcoRI site of TYB1. The plasmid constructed in this manner is called pTYB
1 (FNCBD-FGF).

(F) Preparation of Expression Vector for Hybrid Polypeptide of FNCBD and EGF Plasmid pBS (FNCBD) obtained in (a) above
Of the plasmid pBS (EGF) obtained in (c) above, the DNA fragment of the inserted FNCBD was cut out by KpnI and XhoI (partial digestion) treatment.
It was ligated to the I-SalI site. The thus constructed plasmid incorporates the DNA represented by SEQ ID NO: 16 in the Sequence Listing.
It was named S (FNCBD-EGF).

Nucleotide numbers 1 to 6 of SEQ ID NO: 16 are KpnI recognition sequences for cloning, and nucleotide numbers 5 to 10 are N
coI recognition sequence, base numbers 11 to 12 are frame alignment sequences, base numbers 13 to 18 are NdeI recognition sequences, base numbers 16 to
18 is the start codon sequence, base numbers 19 to 1038 are human FNCB
D cDNA sequence, base numbers 1039 to 1044 are base sequences generated by ligation of the XhoI recognition sequence and SalI recognition sequence, base numbers 1042 to 1056 are base sequences encoding the enterokinase recognition sequence (DDDDK), base number 1057
121215 is the cDNA sequence of EGF, base numbers 1216 to 1218 are stop codons, and base numbers 1219 to 1224 are E.
This is a coRI recognition sequence.

The DNA fragment of the hybrid gene inserted into pBS (FNCBD-EGF) was found to contain NdeI and Ec
It was excised with oRI and ligated to the NdeI-EcoRI site of the expression vector pTYB1.
Plasmid thus constructed was transformed into pTYB1 (FNCBD-
EGF).

(G) Confirmation of expression of hybrid polypeptide of FNCBD and bFGF and hybrid polypeptide of FNCBD and EGF pTYB1 (FNCBD), pTYB1 (FNCBD-FGF) and
In pTYB1 (FNCBD-EGF), E. coli strain ER2566 (NEW EN
GLAND BioLabs). The transformed E. coli was LB containing 100 μg / ml ampicillin.
The cells were cultured overnight at 37 ° C in 2 ml of the medium. 0.02 ml of this preculture was inoculated into 2 ml of SB medium containing 100 μg / ml ampicillin. After culturing until the bacterial turbidity becomes 0.5, IPTG (isopropyl-
(β-D-galactoside) was added, and the cells were further cultured at 37 ° C. for 2 hours and collected.

The whole cell protein was analyzed by SDS-PAGE (1
(2% gel) and Coomassie staining was performed. As a result, in the stained gel, FNCB
D, Hybrid of FNCBD and bFGF and FNCB
Bands were observed at 40 kDa, 57 kDa and 46 kDa corresponding to the molecular weights of the hybrids of D and EGF, respectively, confirming the expression of the recombinant polypeptide.

Separately, the gel after SDS-PAGE was transferred to a nitrocellulose membrane, and a monoclonal antibody specifically recognizing FNCBD (FNC4-4, Takara Shuzo) and a monoclonal antibody specifically recognizing bFGF (FGF-2 ( C-18), Sa
nta Cruz Biotechnology) and a monoclonal antibody that specifically recognizes EGF (Clone10825.1 R & D Systems)
Acted.

As a result, the anti-human FNCBD monoclonal antibody reacted with the 40 kDa polypeptide,
Anti-human FNCBD monoclonal antibody and anti-human bFGF monoclonal antibody react with
It was confirmed that an anti-human FNCBD monoclonal antibody and an anti-human EGF monoclonal antibody reacted with the 6 kDa polypeptide. Thus, the 40 kDa polypeptide is human FNCBD and the 57 kDa polypeptide is human FNCBD.
The hybrid polypeptide of NCBD and human bFGF (FNCBD-FGF) and the 46 kDa polypeptide are considered to be the hybrid polypeptide of human FNCBD and human EGF (FNCBD-EGF).

As described above, the plasmid pTYB1 (FNCB
D), pTYB1 (FNCBD-FGF) and pTYB1 (FNCB
BD-EGF), and E. coli ER2566 (NEW ENGLAND BioLab) in which the expression of each polypeptide was confirmed.
s) was converted to ER2566 [pTYB1 (FNCBD)], ER25
66 [pTYB1 (FNCBD-FGF)], ER2566 [pTYB1 (F
NCBD-EGF)].

(H) Preparation of Expressed Polypeptide The Escherichia coli obtained in (g) above was precultured overnight at 37 ° C. in 2 ml of LB medium containing 100 μg / ml ampicillin. 0.2 ml of this preculture was inoculated into 2 ml of SB medium containing 100 μg / ml ampicillin. IPTG was added, and the cells were further cultured at 37 ° C. and then collected.

The obtained cells were treated with 2 ml of a sonication buffer [50 mM Tris-HCl buffer pH 8.0, 50
mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (E
DTA)], centrifuged and resuspended in 2 ml of the same buffer, and sonicated for 15 seconds and paused for 15 seconds was repeated 6 times on ice to break up the cells.

Triton X-100 (Triton X-100) was added to this suspension to a final concentration of 1%, and the suspension was centrifuged at 5,000 rpm for 20 minutes at 4 ° C. The obtained precipitate was further washed three times with an inclusion body washing solution [0.5% Triton X-100, 1 mM EDTA]. After centrifugation, the insoluble fraction was treated with an 8 M urea solution [8 M urea, 50 mM Tris-HCl buffer pH 8.0, 1
mM EDTA] for 1 hour at room temperature to dissolve.
Next, this lysate was centrifuged at 14,000 rpm for 20 minutes at 4 ° C., and the supernatant was recovered.

The supernatant was dialyzed sequentially against a 4 M urea solution and a 2 M urea solution, and finally, a sonication buffer for a binding activity experiment, or a phosphate buffer [10 mM phosphate buffer] for a cell proliferation activity experiment. Solution pH 8.0, 50 mM sodium chloride].

After dialysis, the mixture was centrifuged at 4 ° C. and 14,000 rpm for 20 minutes, and the supernatant was used as a solubilized recombinant protein sample. In the obtained sample, bands were detected at 40 kDa, 57 kDa and 46 kDa calculated from the amino acid sequence by SDS-PAGE, and the polypeptide as a recombinant protein was confirmed. Furthermore, (b) of Example 2
After -3), a polypeptide sample purified by gelatin sepharose 4B and dialyzed with a phosphate buffer was used.

Example 2 <Examination of Biological Activity> FNCB obtained in Example 1
D, Collagen binding activity and cell proliferation activity were examined using FNCBD-FGF and FNCBD-EGF. (A) Measurement of collagen binding activity The binding activity of FNCBD-FGF to gelatin (Gelatin Sepharose 4B, Amersham Pharmacia Biotech) was examined by a batch method. FIG. 3 shows an outline of the method.

[0139] As a recombinant protein preparation, FNCBD-about 50 µg / ml dialyzed into a sonication buffer was used.
FGF was used. 50 in 1.5 ml microtube
Add 0 μl of FNCBD-FGF and 100 μl of gelatin sepharose 4B turbidity (washed twice with sonication buffer, then made 50% (vol / vol) turbidity) and added 4 ° C.
For 1 hour on a rotator. After centrifugation at 5000 rpm for 30 seconds, the supernatant was recovered. Next, 500 μl of a 1M sodium chloride solution was added to the sediment, and the mixture became turbid.
After centrifugation for 2 seconds, the supernatant was collected. Similarly, the precipitate was washed again with a 1 M sodium chloride solution. Next, use the same operation
Elution of proteins bound to gelatin sepharose 4B was sequentially attempted with M urea, 2 M urea, and 4 M urea solution.
Finally, the protein bound to gelatin sepharose 4B was eluted by heating at 90 ° C. in 100 μl of SDS buffer.

Apart from the above operations, the binding activity of FNCBD-FGF to heparin (AF-Heparintoyopearl, Tosoh) was examined by a batch method. 500 μl of FNCBD-FGF and 1 in a 1.5 ml microtube
00 μl of AF-Heparintoyopearl turbid solution (washed twice with sonication buffer and then made 50% (volume / volume) turbid solution) was added and mixed on a rotator at 4 ° C. for 1 hour. 5,000
After centrifugation at 30 rpm for 30 seconds, the supernatant was recovered. Sinking
After washing twice with 500 μl of 1 M sodium chloride solution (the supernatant was not electrophoresed), the bound protein was eluted by heating at 100 ° C. for 5 minutes in 100 μl of SDS buffer. Each of the collected supernatants (5 μl) was mixed with a 6-fold concentration of SDS buffer (1 μl), allowed to stand at 90 ° C. for 5 minutes, and then subjected to 12% SDS-PAGE.

In the SDS-PAGE gel shown in FIG.
No FNCBD-FGF band was detected in the vicinity of 57 kDa calculated from the amino acid sequence in Lane 3, and FNCBD was detected.
-The binding of FGF to gelatin was revealed. Subsequently, no band was detected by two washes with the 1 M sodium chloride solution (lanes 4 and 5), and it was revealed that FNCBD-FGF did not elute from the gelatin when the 1 M sodium chloride solution was washed. As a result of attempting elution with 1 M urea, 2 M urea, and 4 M urea in the same operation, 1
Almost no band was detected with M urea and 2M urea (lanes 6 and 7), and a faint band was detected with 4M urea (lane 8).

The FNCBD-FGF band was detected by elution with SDS buffer at 90 ° C. (lane 9). That is, for 1 M urea and 2 M urea, FNCBD-F
GF was hardly eluted from gelatin, partially eluted with 4M urea, and mostly eluted with SDS buffer at 90 ° C, confirming the strong binding of FNCBD-FGF to gelatin.

In the binding of FNCBD-FGF to heparin, no band was detected in lane 10, revealing its binding activity. After washing with a 1M sodium chloride solution, heparin is separated from FNC with 90 ° C. SDS buffer.
BD-FGF was eluted and a band was detected (lane 1
1) The binding of FNCBD-FGF to heparin was supported. That is, the bFGF portion has the same three-dimensional structure as natural bFGF, and is retained as a physiological activity.
From the above, it was proved that FNCBD-FGF retained strong gelatin binding in the FNCBD portion, and that the bFGF portion retained heparin binding.

In the enzyme-linked immunosorbent assay (ELISA), bovine gelatin (Sigma), bovine natural collagen (I
-AC, Koken), FNCBD, FNCBD-FGF and FNCBD-E against human telocollagen (I-PC, Koken) and bovine serum albumin (BSA, Sigma)
The binding activity of GF was examined. Figure 5 shows a conceptual diagram of the ELISA method.
Shown in

First, 200 μl / well of a phosphate buffered saline solution of 100 μg / ml of gelatin, natural collagen, atelocollagen, and serum albumin was dispensed into a flat bottom 96-well multiplate for ELISA. It was left at 4 ° C. overnight.

The solution was discarded, and blocked with 3% serum albumin for 24 hours. The wells were washed three times with a 0.05% Tween 20 phosphate buffered saline solution, and then diluted with phosphate buffered saline at 1 μg / ml, 100 nL.
g / ml, 10 ng / ml, 0 ng / ml FNCBD,
FNCBD-FGF and FNCBD-EGF solutions were
μl was dispensed into each well and allowed to stand at 37 ° C. for 1 hour.

After washing three times with a phosphate-buffered saline solution containing 0.05% of Tween 20, 100 μl of anti-human FNCBD monoclonal antibody (Takara Shuzo) diluted to 1/1000 was dispensed. Let stand for hours. 0.05%
Tween 20 / well with phosphate buffered saline solution
After washing twice, a 1000-fold diluted peroxidase-labeled anti-mouse immunoglobulin polyclonal antibody (Dako Japan) was dispensed into 100 μl wells, and allowed to stand at room temperature for 1 hour. After washing the wells 6 times with 0.05% Tween 20 / phosphate buffered saline solution, 0.1 M citrate buffer containing 1 mg / ml o-phenylenediamine and 0.03% hydrogen peroxide Dispense pH 4.7 and add 1
After standing for 0 minutes, the reaction was stopped with 50 μl of 4N sulfuric acid.
The absorbance from 2 nm to 690 nm was measured.

FIG. 6 shows that 100 μg / ml of gelatin, natural collagen, atelocollagen, and serum albumin were respectively immobilized on a multiplate, and 1 μg / ml of FNCBD.
Shows the results obtained when was reacted. The vertical axis indicates absorbance. Absorbance when serum albumin is immobilized is 0.08,
The absorbances of gelatin, natural collagen, and atelocollagen were 2.27, 2.08, and 1.93, respectively, which were more than 10 times that of serum albumin.

FIG. 7 shows that 0.1 μg / ml FNCBD-F
The results of reacting GF with gelatin, natural collagen, atelocollagen, and serum albumin are shown. The absorbances of gelatin, natural collagen, and atelocollagen were 1.60, 2.12, and 2.20, respectively, and the absorbance of serum albumin was 0.27. Thus, FNCBD
-The binding degree of FGF to gelatin, natural collagen, and atelocollagen is significantly higher than that to serum albumin.

Similarly, as shown in FIG. 8, FNCBD-
EGF for serum albumin, gelatin, natural collagen,
As a result of the reaction with atelocollagen, the absorbances were 0.11, 1.54, 1.66, and 1.54, respectively, and the binding to immobilized gelatin, natural collagen, and atelocollagen was clearly higher than the binding to serum albumin.

Although the absorbance in the ELISA method is considered to increase in correlation with the binding activity, the absorbance in FNC is higher than that in the reaction with immobilized serum albumin which can be regarded as non-specific binding.
Reaction with BD and immobilized gelatin, natural collagen, atelocollagen shows about 10 times higher absorbance,
These reactions are considered specific binding reactions (FIG. 6). Similarly, the reaction of FNCBD-FGF shown in FIG. 7 and FNCBD-EGF shown in FIG. 8 with immobilized gelatin and collagen can be said to be specific binding. This means that the polypeptide produced by genetic engineering in the present invention is FNCB.
D shows that the cell growth factor is a hybridized molecule without losing the intrinsic collagen binding activity of D. That is, by using the hybrid polypeptide, it was possible to impart a collagen binding activity to the cell growth factor.

(B) Measurement of Cell Proliferating Activity FNCBD showing collagen binding activity in (a) above,
FNCBD-FGF and FNCBD-EGF were further examined to determine whether they maintained cell proliferative activity.

(B-1) Mouse fibroblast cell line BALB / c3T3 cells (mouse fibroblast cell line)
Suspended in Iscove's modified Dulbecco's Eagle's medium (3% FBS-IMDM) containing 5% fetal bovine serum, dispensed at 2 × 10 ³ cells / well in a 24-well microplate, and then suspended at 37 ° C. in the presence of 5% carbon dioxide. Cultured for hours. Thereafter, the medium was changed to an IMDM medium containing 0.6% fetal bovine serum (0.6% FB).
S-IMDM) and further cultured at 37 ° C. for 24 hours. Next, FNCBD, FNCBD-EGF or EG
F was added to the medium and cultured at 37 ° C. for 4 days. The cells were further cultured at 37 ° C. for 3 hours after the WST-1 reagent (Boehringer Mannheim) was added to 1/10 of the medium. The medium was transferred to a 96-well microplate, and 450 nm-6 was read by a microplate reader.
The absorbance at 90 nm was measured to examine the cell proliferation activity.

As shown in FIG. 9, FNCBD-EGF
Showed a cell growth activity on BALB / c3T3 cells in a concentration-dependent manner, exceeding the cell growth activity of EGF. On the other hand, for FNCBD, BALB / c
It showed cell proliferation activity against 3T3 cells. Therefore, F
By hybridizing NCBD to EGF, EG
It is considered that the cell proliferation activity of F did not disappear. Furthermore, the fact that FNCBD showed cell growth activity and that FNCB
Since D-EGF showed a cell proliferating activity exceeding that of EGF, it is considered that FNCBD-EGF showed a proliferating activity in which FNCBD and EGF were added.

(B-2) Human fibroblasts Human fibroblasts were suspended in Iscove's modified Dulbecco's Eagle's medium (3% FBS-IMDM) containing 3% fetal bovine serum and placed in a 24-well microplate at 5 × 10 ^3. After dispensing cells / well, the cells were cultured at 37 ° C. for 5 hours in the presence of 5% carbon dioxide. Then, the medium was changed to IMD containing 0.6% fetal calf serum.
M medium (0.6% FBS-IMDM)
The cells were cultured at 37 ° C. for 24 hours. Next, FNCBD or FN
CBD-FGF was added and cultured at 37 ° C. for 4 days. X
A TT reagent (Boehringer Mannheim) was added to a half of the medium volume, and the cells were further cultured at 37 ° C for 6 hours. The medium was transferred to a 96-well microplate, and the absorbance at 492 nm to 690 nm was measured using a microplate reader to examine the cell growth activity.

As shown in FIG. 10, FNCBD-FGF
Showed cell growth activity on human fibroblasts in a concentration-dependent manner. On the other hand, for FNCBD, BALB /
Although weaker than the case of c3T3 cells, it showed cell growth activity against human fibroblasts. From the above results, it is considered that the cell growth activity of bFGF was not lost by providing collagen binding to FGF.

(B-3) NRK49F cells Thereafter, a recombinant protein preparation purified from gelatin sepharose 4B and dialyzed against a phosphate buffer was used. NR
K49F cells (rat fibroblast cell line) are suspended in Dulbecco's modified Eagle's medium (2% FBS-DMEM) containing 2% fetal bovine serum, and dispensed at 1 × 10 ⁴ cells / well in a 24-well microplate. And incubated at 37 ° C for 7 hours in the presence of 5% carbon dioxide. Next, FNCBD, FNCBD-
EGF or EGF (n = 3 each) was added to the medium of each well and cultured at 37 ° C. for 4 days. Cells
After adding 1/10 volume of the medium, the WST-1 reagent (Dojindo Laboratories) was cultured at 37 ° C. for 3 hours. The medium was transferred to a 96-well microplate, and read with a microplate reader.
The cell growth activity was determined from the absorbance at nm-690 nm. FIG.
As shown in the figure, NRK49F cells were treated with FNCBD-E
GF showed a cell growth activity in a concentration-dependent manner, and the result was similar to that of EGF. On the other hand, FNCBD did not show cell proliferation activity against NRK49F cells at this concentration. Therefore, it is considered that the cell growth activity of EGF was not lost by hybridizing FNCBD to EGF.

Example 3 <Comparison of Functionality> The functions of collagen-binding cell growth factor and the original cell growth factor were compared. (A) Cell growth activity of cell growth factor-complexed collagen As an example of collagen-binding cell growth factor confirmed to have collagen-binding activity and cell-growth activity, FNCBD-E
After binding GF to collagen (FNCBD-EGF complexed collagen), its cell proliferation activity was examined. This embodiment is schematically shown in FIG. First, 24 for tissue culture
A 3 mg / ml atelocollagen hydrochloride solution (pH 3.0) was dispensed at 500 μl / well into a well microplate and incubated at 4 ° C overnight.
At rest. The solution was discarded, washed four times with a 0.05% Tween 20 phosphate-buffered saline solution, then twice with a phosphate-buffered saline solution, and then once with a serum-free DMEM medium. Next, 0 n diluted in serum-free DMEM medium
M, 2.5 nM, 5 nM, 10 nM, 20 nM, or 40 nM F
An NCBD-EGF or EGF solution (250 μl) was dispensed into each well and allowed to stand at 37 ° C. for 2 hours. Discard solution 0.05% T
The wells were washed twice with a phosphate buffered saline solution (ween 20), then washed six times with a phosphate buffered saline solution, and then washed once with Hank's buffer. Next, bovine pituitary extract as an additive to serum-free human keratinocyte basal medium (KBM2)
Human keratinocytes were suspended in a medium (KBM2 + BPE) supplemented with (BPE) and dispensed into a 24-well microplate at 10 ⁴ cells / 0.5 ml / well. When FNCBD-EGF or EGF was retained at 100% in the collagen coated on the wells, the final concentrations were 0 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM.
M or 20 nM. 5% human keratinocytes under these conditions
The cells were cultured at 37 ° C. for 4 days in the presence of carbon dioxide. And
After adding 1/10 of the medium with WST-1 reagent, the cells are grown for an additional 37
Incubated for 3 hours at ° C. The medium was transferred to a 96-well microplate, and read at 450 nm-69 with a microplate reader.
The absorbance at 0 nm was measured to determine the cell growth activity.

As shown in FIG. 13, unlike EGF, FNCB
D-EGF showed cell growth activity on human keratinocytes in a concentration-dependent manner. It is considered that this result was caused by a difference in collagen binding between FNCBD-EGF and EGF. Therefore,
FNCBD-EGF is a peptide derived from fibronectin and EGF
And a collagen-binding bioactive polypeptide characterized by having both collagen-binding activity and EGF activity.
It has also been found that the polypeptide is a collagen-binding physiologically active polypeptide characterized by exhibiting EGF activity while maintaining the binding or being released slowly after binding to collagen.

The collagen to which FNCBD-EGF is bound is
One of biomaterials (EGF-complexed collagen) characterized by containing collagen-binding EGF and collagen-derived polypeptide. In addition, the above-mentioned method for culturing human keratinocytes is an example of a method for imparting a physiological activity such as promoting a proliferation activity to cells by using a physiologically active peptide-conjugated collagen.

(B) Comparison of binding activity between FNCBD-EGF and EGF for collagen Using ELISA method, FNCBD-EGF and EGF for collagen were used.
Were compared for their binding activity. First, a flat bottom 96 w
3 mg / ml I, II, III or I in ell multiplate
Hydrochloric acid solution of type V atelocollagen (pH3.0) or BSA
Alternatively, Block Ace (blocking agent) was dispensed at 200 μl / well, and allowed to stand at 4 ° C. overnight. Discard the solution and add 0.
5% Tween 20 in phosphate buffered saline
After washing the wells, 0 nM diluted in serum-free DMEM medium, 1.
25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM FNCBD-EGF
Solution or 0 nM, 1.25 nM, 2.5 nM, 5 nM, 10 n
100 μl of M, 20 nM EGF solution was dispensed into each well and allowed to stand at 37 ° C. for 2 hours. Next, after washing three times with 0.05% Tween 20 / phosphate buffered saline solution, the stock solution was
10-fold diluted anti-human EGF monoclonal antibody
0 μl was dispensed and allowed to stand at room temperature for 1 hour. Followed by 0.05%
After washing the wells three times with Tween 20 / phosphate-buffered saline solution, 100 μl of a 1000-fold diluted peroxidase-labeled anti-mouse immunoglobulin polyclonal antibody was dispensed into the wells and allowed to stand at room temperature for 1 hour. Finally 0.05% Twe
The wells were washed six times with an en 20 / phosphate buffered saline solution and then 1 mg / ml o-phenylenediamine (o-phenydiene).
lenediamine) and 0.1M citrate buffer (pH 4.7) containing 0.03% hydrogen peroxide.Dispense for about 10 minutes, and then add 4N sulfuric acid 50μ
The reaction was stopped with l. 492 nm-
The absorbance at 690 nm was measured.

FIG. 14 is a graph showing the obtained absorbance values. The vertical axis indicates collagen binding activity by absorbance, and FNCBD-
The horizontal axis represents EGF or the concentration of EGF. FNCBD-EGF was added to 1.25 nM, 2.5 nM, 5 nM, and 10 nM in wells coated with any of type I, II, III, or type IV atelocollagen.
The absorbance values of wells dispensed at concentrations of M and 20 nM are FN
Assuming that the absorbance of the well into which CBD-EGF was dispensed at 0 nM is 0,
High values were shown in a concentration-dependent manner. On the other hand, EGF was 1.25 nM,
The absorbance of the wells dispensed at the concentrations of 2.5 nM, 5 nM, 10 nM, and 20 nM was almost the same as the well dispensed at the concentration of 0 nM. Since the absorbance in the ELISA method is considered to increase in relation to the binding activity, FNCBD-EGF obtained by fusing EGF with the collagen-binding domain of fibronectin
Showed a remarkable collagen binding property as compared with the original EGF. Therefore, the activity of FNCBD-EGF to promote the proliferation of human keratinocytes shown in (a) of Example 3 was supported based on its collagen binding property. That is,
FNCBD-EGF was retained via collagen and was able to show local cell proliferation activity. The above examples mean that the functions of local retention, sustained release, and targeting to collagen, which were impossible with the original bioactive peptide, were imparted by the present invention while retaining the bioactivity of the bioactive peptide. .

(C) Long-term stability of cell growth activity of cell growth factor-complexed collagen As an example of a collagen-binding cell growth factor confirmed to have collagen binding activity and cell growth activity, FNCBD-EGF was bound to collagen ( FNCBD-EGF complexed collagen) and its cell proliferation activity were examined. First, a 1 mg / ml atelocollagen hydrochloride solution (pH 3.0) was dispensed at 500 μl / well into a 24-well microplate for tissue culture, and allowed to stand at 4 ° C. overnight. The solution was discarded and washed four times with a 0.05% Tween 20 phosphate-buffered saline solution, then twice with a phosphate-buffered saline solution, and once with a serum-free IMDM medium. Next, 0 nM diluted in serum-free DMEM medium,
8 nM, 40 nM, 200 nM or 1000 nM FNCBD-EGF solution
250 μl of (n = 3) was dispensed into each well and allowed to stand at 37 ° C. for 2 hours. The solution was discarded, washed twice with a phosphate buffered saline solution, and then once with DMEM. 500 μl / DMEM
The mixture was dispensed in a well and allowed to stand at 37 ° C. for one week. Next, serum-free
0 pM, 8 pM, 40 pM, 200 pM or 10 pM diluted in DMEM medium
250 μl of a 00 pM EGF solution (n = 2) was dispensed into each well and allowed to stand at 37 ° C. for 2 hours. Discard solution and FNCBD
-Well washed with EGF or EGF twice with 0.05% Tween 20 in phosphate-buffered saline solution, washed 6 times with phosphate-buffered saline solution, and washed once with DMEM did. NRK49F rat fibroblasts were suspended in DMEM medium containing 2% fetal bovine serum, and 10
Dispensed at ⁴ cells / 0.5 ml / well. 100% FNCBD-EGF or EGF in collagen coated on wells
If retained, the final concentration will be 0 pM, 4 pM, 20 pM, 100 pM or 500 pM. Under these conditions, NRK49F cells were cultured at 37 ° C. for 4 days in the presence of 5% carbon dioxide. And WST-1
After addition of one-tenth volume of the medium, the cells were further incubated at 37 ° C for 3 times.
Cultured for hours. Transfer the medium to a 96-well microplate,
The cell proliferation activity was determined by measuring the absorbance at 450 nm-690 nm using a microplate reader.

As shown in FIG. 15, unlike EGF, FNCB
D-EGF showed cell proliferation activity on NRK49F cells in a concentration-dependent manner. It is considered that this result was caused by a difference in collagen binding between FNCBD-EGF and EGF. In addition, FNCBD-EGF maintains both collagen binding activity and EGF activity despite being left at 37 ° C for one week, and after binding to collagen, the binding is maintained or released slowly. It was found to show EGF activity. This result shows that even in the living body, FN
CBD-EGF binds or cells as a biomaterial (EGF complexed collagen) consisting of collagen material,
It means that a biological activity such as proliferation, differentiation, regeneration or promotion of substance production can be imparted to a tissue or organ.

(D) Comparison of collagen targeting activity between FNCBD-EGF and EGF The targeting activity of FNCBD-EGF and EGF to collagen in the presence of a high concentration of contaminating proteins was compared using ELISA. First, 200 μl / well of a 3 mg / ml type I atelocollagen hydrochloric acid solution (pH 3.0) was dispensed into a 96-well flat plate for tissue culture at a temperature of 4 ° C. overnight. The solution was discarded, and the wells were washed 6 times with a 0.05% / Tween20 phosphate buffered saline solution.
100 μl of 0 nM, 2 nM, 4 nM, 8 nM FNCBD-EGF solution or 0 nM, 2 nM, 4 nM, 8 nM EGF solution diluted with DMEM medium solution of high concentration bovine serum albumin (BSA)
l, dispensed into each well and allowed to stand at 37 ° C for 1 hour.
Then, after washing three times with 0.05% Tween 20 / phosphate-buffered saline solution, the stock solution was diluted to 1/1000 with anti-human EGF.
100 μl of the monoclonal antibody was dispensed and allowed to stand at room temperature for 1 hour. Subsequently, the wells were washed three times with 0.05% Tween 20 / phosphate-buffered physiological saline solution, and then 100 μl of a 1000-fold diluted peroxidase-labeled anti-mouse immunoglobulin polyclonal antibody was dispensed into the wells and allowed to stand at room temperature for 1 hour. I let it. Finally, the wells are washed six times with 0.05% Tween 20 / phosphate-buffered saline solution, and then 0.1 M citrate buffer containing 1 mg / ml o-phenylenediamine and 0.03% hydrogen peroxide. After pH4.7 was dispensed and allowed to stand for about 10 minutes, the reaction was stopped with 50 μl of 4N sulfuric acid. The absorbance at 492 nm to 690 nm was measured using a plate reader.

FIG. 16 is a graph showing the obtained absorbance values. The vertical axis indicates the collagen targeting activity by absorbance, and the horizontal axis indicates the concentration of FNCBD-EGF or EGF. FNCBD-EGF in wells coated with type I atelocollagen
The absorbance values of the wells dispensed at the concentrations of nM, 2 nM, 4 nM, and 8 nM showed high values in a concentration-dependent manner. On the other hand, EGF
The absorbance of the wells dispensed at the concentrations of 0 nM, 2 nM, 4 nM, and 8 nM was almost the same as the well dispensed at the concentration of 0 nM. Since the absorbance in the ELISA method is considered to increase in relation to the collagen targeting activity,
It was revealed that FNCBD-EGF obtained by fusing F with the collagen-binding domain of fibronectin exhibited a remarkable collagen targeting activity in the presence of a high concentration of contaminating protein as compared with the original EGF. That is, FNCBD-EGF can specifically target collagen in vivo or in vitro or in the presence of a high concentration of contaminating proteins, and can exhibit local cell growth activity. The above examples are examples in which the functions of local retention and targeting to collagen, which were impossible with the original bioactive peptide, were imparted by the present invention while maintaining the bioactivity of the bioactive peptide. In addition, the present invention was proved to be a novel drug delivery system for bioactive peptides.

(E) Slow release and release characteristics of FNCBD-EGF by plasma FN FNCBD-E from collagen by plasma FN using ELISA method
The release and release characteristics of GF were studied. First, 3 mg / ml I or IV in a 96 well flat bottom tissue culture multiplate
Hydrochloric acid solution of type atelocollagen (pH 3.0) was dispensed at 200 μl / well and left overnight at 4 ° C. The solution was discarded, and the wells were washed 6 times with a 0.05% / Tween 20 phosphate buffered saline solution, and then diluted with a DMEM medium solution.
100 μl of an nM FNCBD-EGF solution was dispensed into each well and allowed to stand at 37 ° C. for 90 minutes. After washing three times with 0.05% Tween 20 / phosphate-buffered physiological saline solution, diluted with PBS solution, FN solution of 20, 40, 80, 160, 320, 640 or 1280 nM concentration or the same weight concentration as FN. (Μg / ml) 100 μl of the diluted series of BSA was dispensed into each well and incubated at 37 ° C.
It was left still for 90 minutes. After washing three times with 0.05% Tween 20 / phosphate-buffered saline solution, 100 μl of the anti-human EGF monoclonal antibody diluted to 1/1000 of the stock solution was dispensed and allowed to stand at room temperature for 1 hour. Subsequently, the wells were washed three times with a 0.05% Tween 20 / phosphate-buffered saline solution, and 100 μl of a 1000-fold diluted peroxidase-labeled anti-mouse immunoglobulin polyclonal antibody was dispensed into the wells.
Let stand for hours. Finally, the wells were washed six times with 0.05% Tween 20 / phosphate-buffered saline solution, and then 1 mg / ml
O-phenylenediamine and 0.
A 0.1 M citrate buffer (pH 4.7) containing 03% hydrogen peroxide was dispensed, allowed to stand for about 10 minutes, and then stopped with 50 μl of 4N sulfuric acid. The absorbance at 492 nm to 690 nm was measured using a plate reader.

FIG. 17 is a graph showing the obtained absorbance values. The vertical axis shows the absorbance of collagen binding activity, and FNCBD-
The molar concentration ratio between EGF and FN is shown on the horizontal axis. After binding FNCBD-EGF to wells coated with type I atelocollagen,
, 40, 80, 160, 320, 640, and 1280 nM, the absorbance values of wells into which FN was dispensed decreased in a FN concentration-dependent manner. Since the decrease in absorbance in the ELISA method is considered to be lower in correlation with the decrease in FNCBD-EGF bound to collagen, FNCBD-EGF in which EGF is fused with the collagen-binding domain of fibronectin is not bound by FN. Was found to be competitively inhibited. That is, it has been revealed that FNCBD-EGF exhibits the property of being specifically and slowly released and released by plasma FN after binding to collagen in vivo or in vitro. The above examples show that invasion, exposure, addition or coexistence of plasma, serum or blood, collagen-binding bioactive polypeptide is slowly released from collagen,
Alternatively, it is an example having a feature that collagen is retained in a site where blood is insufficient, that is, a site where a bioactive peptide such as a cell growth factor is starved. Therefore, the present invention has been proved to be a novel drug delivery system that targets a bioactive peptide to collagen, is retained, and performs controlled sustained release.

[0169]

Industrial Applicability The collagen-binding bioactive polypeptide to which the activity of the bioactive peptide is maintained and the binding activity to collagen is imparted is useful as a drug delivery system (DDS) for the bioactive peptide.
Further, the polypeptide can be complexed with collagen, and the collagen matrix thus functionally modified is useful as a new biomaterial for tissue regeneration.

[0170]

[Sequence Listing Free Text] SEQ ID NO: 1 Description of artificial sequence: Amino acid sequence of modified human fibronectin collagen-binding domain (2) .. (341): / note = "human fibronectin collagen-binding domain" SEQ ID NO: 2. Description of artificial sequence: Amino acid sequence of human fibroblast growth factor to which enterokinase recognition sequence is added (1) .. (5): / note = "enterokinase recognition sequence" (6) .. (159): / Note = "Human fibroblast growth factor" SEQ ID NO: 3 Description of artificial sequence: Amino acid sequence of human epidermal growth factor with added enterokinase recognition sequence (1) .. (5): / Note = "Enterokinase recognition Sequence "(6) .. (58): / Note =" Human epidermal growth factor "SEQ ID NO: 4 Description of artificial sequence: Amino acid sequence of hybrid polypeptide consisting of human fibronectin collagen-binding domain and human fibroblast growth factor (2) .. (341): / note = "human fibronectin collagen-binding domain" (343) .. (347): / note = "enterokinase recognition sequence" (348) .. (501): / note "Human fibroblast growth factor" SEQ ID NO: 5 Description of artificial sequence: Amino acid sequence of hybrid polypeptide consisting of human fibronectin collagen-binding domain and human epidermal growth factor (2) .. (341): / note = " Human fibronectin collagen binding domain "(343) .. (347): / note =" enterokinase recognition sequence "(348) .. (400): / note =" human epidermal growth factor "SEQ ID NO: 6 Description: PCR sense primer for human fibronectin collagen binding domain SEQ ID NO: 7 Description of artificial sequence: PCR antisense primer for human fibronectin collagen binding domain SEQ ID NO: 8 Description of artificial sequence: Modified human phyllase SEQ ID NO: 9 Artificial sequence description: PCR sense primer for human fibroblast growth factor SEQ ID NO: 10 Artificial sequence description: PCR antisense primer for human fibroblast growth factor SEQ ID NO: 11 Description of artificial sequence: Nucleotide sequence of DNA encoding human fibroblast growth factor to which an enterokinase recognition sequence has been added (228): / note = "point mutation caused by polymerase chain reaction" SEQ ID NO: 12 artificial Description of sequence: PCR sense primer of human epidermal growth factor SEQ ID NO: 13 Description of artificial sequence: PCR antisense primer of human epidermal growth factor SEQ ID NO: 14 Description of artificial sequence: Description of artificial epidermal growth factor to which an enterokinase recognition sequence is added Base sequence of DNA to encode SEQ ID NO: 15 artificial Column description: Nucleotide sequence encoding a hybrid polypeptide consisting of human fibronectin collagen-binding domain and human fibroblast growth factor SEQ ID NO: 16 Description of artificial sequence: Human fibronectin collagen-binding domain and human epidermal growth factor Sequence of DNA encoding hybrid polypeptide consisting of

[0171]

[Sequence List] SEQUENCE LISTING <110> Terumo Corporation <120> Functional Hybrid Polypeptide with Collagen-binding Activity <130> 19990120 <140> <141> <160> 16 <170> PatentIn Ver. 2.0 <210> 1 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified Human Fibronectin Collagen-Binding Domain <220> <221> INIT _MET <222> (1) <220> <221> DOMAIN < 222> (2) .. (341) <223> / note = "human fibronectin collagen-binding domain" <220> <221> CONFLICT <222> (69) <220> <221> CONFLICT <222> (125) <400> 1 Met Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro Tyr 1 5 10 15 Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met Gln 20 25 30 Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu Gly 35 40 45 Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly Gly 50 55 60 Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly Arg 65 70 75 80 Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu Trp 85 90 95 Cys Ser Thr Thr Ser As n Tyr Glu Gln Asp Gln Lys Tyr Ser Phe Cys 100 105 110 Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn Gly 115 120 125 Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr Asp 130 135 140 Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr Thr 145 150 155 160 Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala Ala 165 170 175 His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile Gly 180 185 190 Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys Thr 195 200 205 Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser Gln 210 215 220 Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn Asp 225 230 235 240 Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr Cys 245 250 255 Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln Cys 260 265 270 Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp Glu 275 280 285 Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg Gly 290 295 300 Ile Gly Glu Trp His Cys Gl n Pro Leu Gln Thr Tyr Pro Ser Ser Ser 305 310 315 320 Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn Ser 325 330 335 His Pro Ile Gln Trp Leu Glu 340 <210> 2 <211> 159 < 212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human Basic Fibroblast Growth Factor with Enterokinase Recognition Sequence <220> <221> PEPTIDE <222> (1) .. (5) <223> / note = "enterokinase recognition sequence" <220> <221> PEPTIDE <222> (6) .. (159) <223> / note = "human fibroblast growth factor" <400> 2 Asp Asp Asp Asp Lys Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu 1 5 10 15 Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp 20 25 30 Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His 35 40 45 Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile 50 55 60 Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly 65 70 75 80 Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu 85 90 95 Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Ph e Glu Arg Leu Glu 100 105 110 Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr 115 120 125 Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly 130 135 140 Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 <210> 3 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human Epidermal Growth Factor with Enterokinase Recognition Sequence <220> <221> PEPTIDE <222> (1) .. (5) <223> / note = "enterokinase recognition sequence" <220> <221> PEPTIDE <222> (6) .. (58) <223> / note = "human epidermal growth factor" <400> 3 Asp Asp Asp Asp Lys Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp 1 5 10 15 Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp 20 25 30 Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln 35 40 45 Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 50 55 <210> 4 <211> 501 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hybrid Polypeptide of Human Fibronectin Collagen-Binding Domain and Human Basic Fibroblast Growth Factor <220> <221> INIT MET <222> (1) <220> <221> DOMAIN <222> (2) .. (341) <223> / note = "human fibronectin collagen-binding domain "<220> <221> PEPTIDE <222> (343) .. (347) <223> / note =" enterokinase recognition sequence "<220> <221> PEPTIDE <222> (348) .. (501) <223> / note = "human fibroblast growth factor" <400> 4 Met Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro Tyr 1 5 10 15 Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met Gln 20 25 30 Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu Gly 35 40 45 Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly Gly 50 55 60 Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly Arg 65 70 75 80 Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu Trp 85 90 95 Cys Ser Thr Thr Ser Sern Tyr Glu Gln Asp Gln Lys Tyr Ser Phe Cys 100 105 110 Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn Gly 115 120 125 Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr Asp 130 135 140 Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr Thr 145 150 155 160 Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala Ala 165 170 175 His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile Gly 180 185 190 Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys Thr 195 200 205 Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser Gln 210 215 220 Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn Asp 225 230 235 240 Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr Cys 245 250 255 Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln Cys 260 265 270 Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp Glu 275 280 285 Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg Gly 290 295 300 Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser Ser 305 310 315 320 Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn Ser 325 330 335 His Pro Ile Gln Trp Leu Asp Asp Asp Asp Lys Ala Ala Gly Ser Ile 340 345 350 Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro 355 360 365 Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly 370 375 380 380 Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu 385 390 395 400 Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly 405 410 415 Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys 420 425 430 Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe 435 440 445 Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg 450 455 460 Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys 465 470 475 480 480 Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro 485 490 495 Met Ser Ala Lys Ser 500 <210> 5 <211> 400 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hybrid Polypeptide of Human Fibronectin Collagen-Binding Domain and Human Epidermal Growth Factor <220> <221> INIT _MET <222> (1) <220> <221 > DOMAIN <222> (2) .. (341) <223> / note = "human fibronectin collagen-binding domain" <220> <221> PEPTIDE <222> (343) .. (347) <223> / note = "enterokinase recognition sequence" <220> <221> PEPTIDE <222> (348) .. (400) <223> / note = "human epidermal growth factor" <400> 5 Met Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro Tyr 1 5 10 15 Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met Gln 20 25 30 Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu Gly 35 40 45 Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly Gly 50 55 60 Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly Arg 65 70 75 80 Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu Trp 85 90 95 Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe Cys 100 105 110 Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn Gly 115 120 125 Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr Asp 130 135 140 Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr Thr 145 1 50 155 160 Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala Ala 165 170 175 His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile Gly 180 185 190 Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys Thr 195 200 205 Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser Gln 210 215 220 Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn Asp 225 230 235 240 Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr Cys 245 250 255 Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln Cys 260 265 270 Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp Glu 275 280 285 Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg Gly 290 295 300 Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser Ser 305 310 310 315 320 Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn Ser 325 330 335 His Pro Ile Gln Trp Leu Asp Asp Asp Asp Lys Asn Ser Asp Ser Glu 340 345 350 Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met 355 Three 60 365 Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr 370 375 380 Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 385 390 395 400 <210> 6 <211> 49 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Sense Primer for Human Fibronectin Collagen-Binding Domain <400> 6 gaggtaccat ggtacatatg gcagctgttt accaaccgca gcctcaccc 49 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Antisense Primer for Human Fibronectin Collagen-Binding Domain <220> <400> 7 cgggatcctt actcgagcca ctggatgggg tgggagttgg gctgac 46 <210> 8 <211> 1053 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified Human Fibronectin Collagen-Binding Domain <220> <221> conflict <222> (109) <220> <221> conflict <222> (206 ) <220> <221> conflict <222> (270) <220> <221> conflict <222> (374) <220> <221> conflict <222> (681) <400> 8 ggtaccatgg tacatatggc agctgtttac caaccgcagc ct caccccca gcctcctccc 60 tatggccact gtgtcacaga cagtggtgtg gtctactctg tggggatgca gtggctgaag 120 acacaaggaa ataagcaaat gctttgcacg tgcctgggca acggagtcag ctgccaagag 180 acagctgtaa cccagactta cggtggcaac tcaaatggag agccatgtgt cttaccattc 240 acctacaatg gcaggacgtt ctactcctgc accacagaag ggcgacagga cggacatctt 300 tggtgcagca caacttcgaa ttatgagcag gaccagaaat actctttctg cacagaccac 360 actgttttgg ttcagactcg aggaggaaat tccaatggtg ccttgtgcca cttccccttc 420 ctatacaaca accacaatta cactgattgc acttctgagg gcagaagaga caacatgaag 480 tggtgtggga ccacacagaa ctatgatgcc gaccagaagt ttgggttctg ccccatggct 540 gcccacgagg aaatctgcac aaccaatgaa ggggtcatgt accgcattgg agatcagtgg 600 gataagcagc atgacatggg tcacatgatg aggtgcacgt gtgttgggaa tggtcgtggg 660 gaatggacat gcattgccta ctcgcagctt cgagatcagt gcattgttga tgacatcact 720 tacaatgtga acgacacatt ccacaagcgt catgaagagg ggcacatgct gaactgtaca 780 tgcttcggtc agggtcgggg caggtggaag tgtgatcccg tcgaccaatg ccaggattca 840 gagactggga cgttttatca aattggagat tcatgggaga agtatgtgca tggtgtcaga 9 00 taccagtgct actgctatgg ccgtggcatt ggggagtggc attgccaacc tttacagacc 960 tatccaagct caagtggtcc tgtcgaagta tttatcactg agactccgag tcagcccaac 1020 tcccacccca tccagtggct cgagtaagnial 1202 <10> <c> Analyzer> Primer for Human Basic Fibroblast Growth Factor <400> 9 gagtcgacga cgatgataag gcagccggga gcatcaccac 40 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Antisense Primer for Human Basic Fibroblast Growth Factor <400> 10 ggaattctca gctcttagca gacattggaa g 31 <210> 11 <211> 489 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human Basic Fibroblast Growth Factor with Enterokinase Recognition Sequence < 220> <221> mutation <222> (228) <223> / note = "mutation caused by polymerase chain reaction" <400> 11 gtcgacgacg atgataaggc agccgggagc atcaccacgc tgcccgcctt gcccgaggat 60 ggcggcagcg gcgccttccc gcccggccac ttcaaggac c ccaagcggct gtactgcaaa 120 aacgggggct tcttcctgcg catccacccc gacggccgag ttgacggggt ccgggagaag 180 agcgaccctc acatcaagct acaacttcaa gcagaagaga gaggagtcgt gtctatcaaa 240 ggagtgtgtg ctaaccgtta cctggctatg aaggaagatg gaagattact ggcttctaaa 300 tgtgttacgg atgagtgttt cttttttgaa cgattggaat ctaataacta caatacttac 360 cggtcaagga aatacaccag ttggtatgtg gcactgaaac gaactgggca gtataaactt 420 ggatccaaaa caggacctgg gcagaaagct atactttttc ttccaatgtc tgctaagagc 480 tgagaattc 489 <210 > 12 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Sense Primer for Human Epidermal Growth Factor <400> 12 gtgtcgacga cgatgataag aatagtgact ctgaatgtcc cctg 44 <210> 13 <211 > 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Antisense Primer for Human Epidermal Growth Factor <400> 13 gaattcttag cgcagttccc accacttcag 30 <210> 14 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Human Epidermal Growt h Factor with Enterokinase Recognition Sequence <400> 14 gtcgacgacg atgataagaa tagtgactct gaatgtcccc tgtcccacga tgggtactgc 60 ctccatgatg gtgtgtgcat gtatattgaa gcattggaca agtatgcatg caactgtgtt 120 gttggctaca tcggggagcg atgtcagtac cgagacctga agtggtggga actgcgctaa 180 gaattc 186 <210> 15 <211> 1527 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hybrid Polypeptide of Human Fibronectin Collagen-Binding Domain and Human Fibroblast Growth Factor <400> 15 ggtaccatgg tacatatggc agctgtttac caaccgcagc ctcaccccca gcctcctccc 60 tatggccact gtgtcacaga cagtggtgtg gtctactctg tggggatgca gtggctgaag 120 acacaaggaa ataagcaaat gctttgcacg tgcctgggca acggagtcag ctgccaagag 180 acagctgtaa cccagactta cggtggcaac tcaaatggag agccatgtgt cttaccattc 240 acctacaatg gcaggacgtt ctactcctgc accacagaag ggcgacagga cggacatctt 300 tggtgcagca caacttcgaa ttatgagcag gaccagaaat actctttctg cacagaccac 360 actgttttgg ttcagactcg aggaggaaat tccaatggtg ccttgtgcca cttccccttc 420 ctatacaaca accacaatta c actgattgc acttctgagg gcagaagaga caacatgaag 480 tggtgtggga ccacacagaa ctatgatgcc gaccagaagt ttgggttctg ccccatggct 540 gcccacgagg aaatctgcac aaccaatgaa ggggtcatgt accgcattgg agatcagtgg 600 gataagcagc atgacatggg tcacatgatg aggtgcacgt gtgttgggaa tggtcgtggg 660 gaatggacat gcattgccta ctcgcagctt cgagatcagt gcattgttga tgacatcact 720 tacaatgtga acgacacatt ccacaagcgt catgaagagg ggcacatgct gaactgtaca 780 tgcttcggtc agggtcgggg caggtggaag tgtgatcccg tcgaccaatg ccaggattca 840 gagactggga cgttttatca aattggagat tcatgggaga agtatgtgca tggtgtcaga 900 taccagtgct actgctatgg ccgtggcatt ggggagtggc attgccaacc tttacagacc 960 tatccaagct caagtggtcc tgtcgaagta tttatcactg agactccgag tcagcccaac 1020 tcccacccca tccagtggct cgacgacgat gataaggcag ccgggagcat caccacgctg 1080 cccgccttgc ccgaggatgg cggcagcggc gccttcccgc ccggccactt caaggacccc 1140 aagcggctgt actgcaaaaa cgggggcttc ttcctgcgca tccaccccga cggccgagtt 1200 gacggggtcc gggagaagag cgaccctcac atcaagctac aacttcaagc agaagagaga 1260 ggagtcgtgt ctatcaaagg agtgtgtgct aaccg ttacc tggctatgaa ggaagatgga 1320 agattactgg cttctaaatg tgttacggat gagtgtttct tttttgaacg attggaatct 1380 aataactaca atacttaccg gtcaaggaaa tacaccagtt ggtatgtggc actgaaacga 1440 actgggcagt ataaacttgg atccaaaaca ggacctgggc agaaagctat actttttctt 1500 ccaatgtctg ctaagagctg agaattc 1527 <210> 16 <211> 1224 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Hybrid Polypeptide of Human Fibronectin Collagen-Binding Domain and Human Epidermal growth factor <400> 16 ggtaccatgg tacatatggc agctgtttac caaccgcagc ctcaccccca gcctcctccc 60 tatggccact gtgtcacaga cagtggtgtg gtctactctg tggggatgca gtggctgaag 120 acacaaggaa ataagcaaat gctttgcacg tgcctgggca acggagtcag ctgccaagag 180 acagctgtaa cccagactta cggtggcaac tcaaatggag agccatgtgt cttaccattc 240 acctacaatg gcaggacgtt ctactcctgc accacagaag ggcgacagga cggacatctt 300 tggtgcagca caacttcgaa ttatgagcag gaccagaaat actctttttg cacagaccac ttcagactcg ccc gag ccct ag cg cg ccc gag ta cactgattgc acttctgagg gcagaagaga caacatgaag 480 tggtgtggga ccacacagaa ctatgatgcc gaccagaagt ttgggttctg ccccatggct 540 gcccacgagg aaatctgcac aaccaatgaa ggggtcatgt accgcattgg agatcagtgg 600 gataagcagc atgacatggg tcacatgatg aggtgcacgt gtgttgggaa tggtcgtggg 660 gaatggacat gcattgccta ctcgcagctt cgagatcagt gcattgttga tgacatcact 720 tacaatgtga acgacacatt ccacaagcgt catgaagagg ggcacatgct gaactgtaca 780 tgcttcggtc agggtcgggg caggtggaag tgtgatcccg tcgaccaatg ccaggattca 840 gagactggga cgttttatca aattggagat tcatgggaga agtatgtgca tggtgtcaga 900 taccagtgct actgctatgg ccgtggcatt ggggagtggc attgccaacc tttacagacc 960 tatccaagct caagtggtcc tgtcgaagta tttatcactg agactccgag tcagcccaac 1020 tcccacccca tccagtggct cgacgacgat gataagaata gtgactctga atgtcccctg 1080 tcccacgatg ggtactgcct ccatgatggt gtgtgcatgt atattgaagc attggacaag 1140 tatgcatgca actgtgttgt tggctacatc ggggagcgat gtcagtaccg agacctgaag 1200 tggtgggaac tgcgctaaga attc 1224

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a collagen-binding physiologically active polypeptide.

FIG. 2 is a schematic diagram of a bioactive peptide-complexed collagen matrix.

FIG. 3 is a conceptual diagram of a batch method for examining gelatin binding properties of FNCBD-FGF.

FIG. 4 is a view showing a SDS-PAGE gel of a supernatant in a batch method.

FIG. 5 is a conceptual diagram of an ELISA for examining collagen binding activity.

FIG. 6. Collagen binding activity of FNCBD (ELIS
FIG.

FIG. 7 shows the collagen binding activity (ELISA) of FNCBD-FGF.

FIG. 8 shows the collagen binding activity (ELISA) of FNCBD-EGF.

FIG. 9 is a graph showing the proliferative activity of FNCBD-EGF on BALB / c3T3 cells.

FIG. 10 shows the proliferative activity of FNCBD-FGF on human fibroblasts.

FIG. 11 is a graph showing the cell proliferation activity of FNCBD-EGF on NRK49F cells.

FIG. 12 is a diagram schematically showing the novel functionality of a collagen-binding physiologically active polypeptide.

FIG. 13. FNCBD-EGF complexed collagen and E
It is a figure which shows the proliferation activity with respect to human keratinocytes of GF.

FIG. 14 is a diagram showing a comparison of collagen binding activity between FNCBD-EGF and EGF.

FIG. 15 is a graph showing long-term stability of cell growth activity by FNCBD-EGF-conjugated collagen.

FIG. 16: FNCBD- in high concentration protein solution
FIG. 3 is a diagram showing a comparison of collagen targeting activity between EGF and EGF.

FIG. 17 is a graph showing sustained release and release characteristics of FNCBD-EGF by human plasma FN.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21 / 02 C 5/10 H // C12P 21/02 C12R 1:19) C12N 15/00 ZNAA (C12N 15/09 ZNA 5/00 A C12R 1:19) C12R 1:19) F term (reference) 4B024 AA01 BA03 BA21 CA07 DA01 DA02 DA05 DA11 EA04 GA11 HA01 4B064 AG02 AG13 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C076 AA95 CC41 CC50 EE43 FF32 4H045 AA10 BA10 FA41 DA40

Claims (15)

[Claims] 1. A polypeptide comprising a fibronectin-derived peptide and a physiologically active peptide linked to each other, wherein the collagen-binding physiologically active polypeptide has both collagen-binding activity and physiological activity. 2. The collagen-binding bioactive polypeptide according to claim 1, wherein the bioactive peptide is linked to the carboxyl terminus of a fibronectin-derived peptide. 3. The fibronectin-derived peptide according to claim 1,
An amino acid sequence obtained by protease degradation of fibronectin and constituting a collagen binding domain,
Alternatively, the collagen-binding bioactive polypeptide according to claim 1 or 2, comprising a collagen-binding amino acid sequence homologous to the sequence or having one or several amino acids deleted, substituted, inserted or added. 4. The fibronectin-derived peptide has a deletion, substitution, or insertion of an amino acid sequence constituting the polypeptide from Ala ²⁶⁰ to Trp ⁵⁹⁹ of human fibronectin, or homologous or one or several amino acids with the sequence. The collagen-binding physiologically active polypeptide according to claim 1 or 2, which comprises an added amino acid sequence. 5. The collagen-binding bioactive polypeptide according to claim 1, wherein the bioactive peptide is a cytokine or a cell growth factor. 6. The collagen-binding bioactive polypeptide according to claim 1, wherein the bioactive peptide is linked to the fibronectin-derived peptide by a genetic engineering technique. 7. The collagen-binding bioactive polypeptide according to any one of claims 1 to 6, wherein the bioactive peptide is linked to the fibronectin-derived peptide by a genetic engineering technique and is produced in bacteria. 8. The collagen-binding bioactive polypeptide according to claim 1, wherein the collagen-binding bioactive polypeptide is water-soluble. 9. The method according to claim 1, wherein after binding to collagen, physiological activity is exhibited while maintaining the binding or being released gradually.
9. The collagen-binding bioactive polypeptide according to any one of items 1 to 8. 10. A local maintenance agent, sustained release agent or bioactivity imparting agent for a bioactive polypeptide containing the collagen-binding bioactive polypeptide according to any one of claims 1 to 9. 11. A biomaterial comprising a bioactive peptide-complexed collagen in which a collagen-binding bioactive polypeptide and a collagen-derived polypeptide are complexed. 12. The biomaterial according to claim 11, wherein the collagen-binding bioactive polypeptide is the collagen-binding bioactive polypeptide according to any one of claims 1 to 9. 13. A local maintenance agent, sustained release agent or bioactivity imparting agent for a bioactive polypeptide containing the biomaterial according to claim 11 or 12. 14. A recombinant vector containing a gene encoding the collagen-binding bioactive polypeptide according to any one of claims 1 to 9. 15. A transformant comprising a recombinant vector containing a gene encoding the collagen-binding physiologically active polypeptide according to any one of claims 1 to 9.